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PART
VITAMINS
17
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C H A P T E R
155
Inherited Disorders of Folate
and Cobalamin Transport
and Metabolism
David S. Rosenblatt
_
Wayne A. Fenton
1. Folate coenzymes participate in a number of critical
single-carbon transfer reactions, including those involved
in the biosynthesis of pyrimidines, purines, serine, and
methionine and in the degradation of histidine and
purines.
2. Five inherited disorders of folate transport and metabolism have been well substantiated: methylene-H4Folate
reductase de®ciency (MIM 236250); functional methyltetrahydrofolate (methyl-H4Folate):homocysteine methyltransferase (methionine synthase) de®ciency caused by
mutations in the gene for methionine synthase reductase
(cblE) (MIM 236270) or mutations in the gene for
methionine synthase itself (cblG) (MIM 250940); glutamate formiminotransferase de®ciency (MIM 229100); and
hereditary folate malabsorption (MIM 229050).
3. Four putative inherited disorders in the literature cannot
be considered to be well substantiated: dihydrofolate
reductase de®ciency; methenyl-H4Folate cyclohydrolase
de®ciency; cellular uptake defects; and the original
description of primary methyl-H4Folate: homocysteine
methyltransferase de®ciency from Japan.
4. Methylene-H4Folate reductase de®ciency, the most widely
studied of the inherited disorders of folate metabolism, is a
condition in which clinical severity correlates with the
degree of enzyme de®ciency. The clinical symptoms vary,
with developmental delay accompanied by motor and gait
abnormalities, seizures, and psychiatric manifestations
being described. The age of onset has ranged from the
neonatal period to adulthood. The major biochemical
®ndings are moderate homocystinuria and hyperhomocystinemia with low or relatively normal levels of plasma
methionine. Most severely affected patients have died.
Pathologic ®ndings include vascular changes similar to
those seen in classical homocystinuria and demyelination
presumably due to low levels of neurotransmitters or
methionine in the central nervous system. A variant form
of methylene-H4Folate reductase de®ciency resulting in
A list of standard abbreviations is located immediately preceding the index in each
volume. Additional abbreviations used in this chapter include: Ado-B12 or AdoCbl ˆ
50 deoxyadenosylcobalamin; AICAR ˆ 5-phosphoribosyl-5-aminoimidazole-4-carboxamide; Cbl ˆ cobalamin; cbl ˆ cobalamin metabolism locus (cblA, cblB, etc.);
CN-Cbl ˆ cyanocobalamin; FGAR ˆ a-N-formyl-glycinamide ribonucleotide;
FIGLU ˆ formiminoglutamate; GAR ˆ 5-phosphoribosylglycinamide; GSCbl ˆ
glutathionylcobalamin; H2PteGlu or H2Folate ˆ dihydrofolate; H4PteGlu, H4Folate,
or THF ˆ tetrahydrofolate; IF ˆ intrinsic factor; methyl-B12, CH3-B12, or MeCbl ˆ
methylcobalamin; methyl-H4Folate ˆ N 5-methyltetrahydrofolate; mut ˆ methylmalonyl CoA mutase locus; OH-B12 or OH-Cbl ˆ hydroxocobalamin; TC (I, II, or III) ˆ
transcobalamin (I, II, or III).
``intermediate homocystinuria'' is associated with 50
percent residual activity and enzyme thermolability, and
is suggested to be an inherited risk factor for coronary
heart disease. In the majority of cases, this variant is due to
homozygosity for a common polymorphism, 677C!T, in
the methylene-H4Folate reductase gene. Severe methyleneH4Folate reductase de®ciency is resistant to treatment;
folates, methionine, pyridoxine, cobalamin, and carnitine
have all been used. Betaine has the theoretical advantage
of both lowering homocysteine levels and supplementing
methionine levels and has been the most promising
therapeutic agent to date, particularly if started immediately after birth. Nevertheless, the prognosis is generally
poor.
5. Functional methionine synthase de®ciency due to the cblE
and cblG mutations is characterized by homocystinuria
and defective biosynthesis of methionine. Most patients
have presented in the ®rst few months of life with
megaloblastic anemia and developmental delay. At least
one patient presented in early adulthood with a misdiagnosis of multiple sclerosis. The distribution of cobalamin
derivatives was altered in cultured cells, with decreased
levels of MeCbl as compared with normal ®broblasts. The
cblE mutation is associated with low methionine synthase
activity when the assay is performed with low levels of
thiol, whereas the cblG mutation is associated with low
activity under all assay conditions. cblE and cblG
represent distinct complementation classes. Both diseases
respond to treatment with hydroxocobalamin (OH-Cbl).
6. Glutamate formiminotransferase de®ciency is a heterogeneous condition associated with elevated excretion of
formiminoglutamic acid, 4-amino-5-imidazole-carboxamide, and hydantoin-5-propionate. Clinical ®ndings have
varied from mental and physical retardation to massive
excretion of formiminoglutamate in the absence of
retardation. Therapy with folates and methionine has
been described, but given that the correlation between
symptoms and formiminoglutamate excretion remains
uncertain, the basis for treating these patients is unclear.
7. Hereditary folate malabsorption is characterized by the
early onset of failure to thrive and severe folate-responsive
megaloblastic anemia. All patients have been severely
restricted in their ability to absorb oral folic acid or oral
reduced folates. Severe mental retardation may be a
prominent feature if therapy does not succeed in maintaining adequate levels of folate in the cerebrospinal ¯uid.
Two patients have shown increased susceptibility to
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3898
PART 17 / VITAMINS
8.
9.
10.
11.
12.
13.
14.
15.
16.
infection. This disorder provides the best evidence for the
existence of a speci®c carrier for folate both at the level of
the intestine and at the choroid plexus. Therapy has been
attempted with large doses of oral or systemic folates.
All of the clearly delineated disorders of folate metabolism
appear to be inherited as autosomal recessive traits.
Heterozygotes for methylene-H4Folate reductase de®ciency show decreased enzyme levels in somatic cells. A
difference in folate absorption in the heterozygote has been
suggested in at least one family with hereditary folate
malabsorption.
Prenatal diagnosis has been successfully performed for
methylene-H4Folate reductase de®ciency, methionine
synthase reductase de®ciency (cblE), and methionine
synthase (cblG) de®ciency using cultured amniotic cells.
Cobalamins (Cbls) are complex organometallic substances
consisting of a corrin ring, a central cobalt atom, and
various axial ligands. The basic structure, known as
vitamin B12, is synthesized exclusively by microorganisms,
but most higher animals are capable of converting the
vitamin into the two required coenzyme forms, adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl).
Dietary Cbl is acquired mostly from animal sources,
including meat and milk, and is absorbed in a series of
steps that includes proteolytic release from its associated
proteins, binding to a gastric secretory protein known as
intrinsic factor (IF), recognition of the IF-Cbl complex by
cubilin, a receptor on ileal mucosal cells, transport across
those cells, and release into the portal circulation bound to
transcobalamin II (TC II), the serum protein that carries
newly absorbed Cbl throughout the body.
The cellular metabolism by which the coenzymes are
formed involves receptor-mediated binding of the TC IICbl complex to the cell surface, adsorptive endocytosis of
the complex, intralysosomal degradation of the TC II,
release of Cbl into the cytoplasm, enzyme-mediated
reduction of the central cobalt atom, and cytosolic
methylation to form MeCbl or mitochondrial adenosylation to form AdoCbl.
Only two enzymes in mammalian cells are known to
depend on cobalamin coenzymes: methylmalonyl CoA
mutase, which requires AdoCbl; and methionine synthase
(also known as N5-methyltetrahydrofolate:homocysteine
methyltransferase), which requires MeCbl.
Ten different inherited defects are known to impair the
pathways of Cbl transport and metabolism in humans (see
Fig. 155-12). Three affect absorption and transport; the
other seven alter cellular utilization and coenzyme
production.
The defects affecting Cbl absorption and transport
generally manifest themselves in infancy or early childhood as developmental delay with megaloblastic anemia.
Serum Cbl levels may be reduced (in IF (MIM 261000) or
cubilin-protein de®ciency (MIM 261100)) or near normal
(in TC II de®ciency (MIM 275350)). Treatment with
periodic injections of Cbl, with or without folate therapy, is
generally effective in controlling these problems.
The clinical manifestations of de®ciencies in cellular Cbl
utilization and metabolism vary depending on whether one
or both coenzymes are affected. Two abnormalities in
AdoCbl synthesis only (designated cblA (MIM 251100) and
cblB (MIM 251110) lead to impaired methylmalonyl CoA
mutase activity and result in methylmalonic acidemia. In
most, but not all, patients with these defects, pharmacologic supplements of Cbl (cyanocobalamin or hydroxocobalamin) produce distinct reductions in methylmalonate
17.
18.
19.
20.
accumulation and offer a valuable therapeutic adjunct to
dietary protein limitation. Oral antibiotic therapy may be
useful to reduce propionate production by gut bacteria.
The defect in cblA is unknown, while the defect in cblB
patients is in cob(I)alamin adenosyltransferase, the ®nal
step of AdoCbl biosynthesis.
Three distinct mutations, designated cblC (MIM 277400),
cblD (MIM 277410), and cblF (MIM 277380), lead to
impaired synthesis of both AdoCbl and MeCbl and,
accordingly, to de®cient activity of both methylmalonyl
CoA mutase and methionine synthase. Children from these
groups have methylmalonic aciduria and homocystinuria.
Children with the cblC mutation appear to be more
severely affected clinically than the two known sibs in the
cblD group or those in the cblF group. Major clinical
problems in cblC patients include failure to thrive,
developmental retardation, and such hematologic abnormalities as megaloblastic anemia and macrocytosis. Treatment requires a combination of the therapies for the
individual coenzyme de®ciencies: protein restriction and
pharmacologic doses of hydroxocobalamin, possibly in
combination with oral antibiotics and betaine supplements. The precise defects in the cblC and cblD patients
are not yet known, but they must involve early steps in the
intracellular metabolism of cobalamins, possibly cytosolic
Cbl reduction. The defect in cblF appears to be in the
transport mechanism by which Cbl is released from
lysosomes.
The discriminating biochemical features of the inherited
defects in Cbl transport and metabolism are shown in
Table 155-5.
All the disorders of Cbl metabolism for which there are
adequate data are inherited as autosomal recessive traits.
Heterozygotes can be detected only for cblB. Genetic
complementation analyses with somatic-cell heterokaryons have been particularly useful in demonstrating
genetic heterogeneity and in con®rming the existence of
autosomal recessive inheritance among defects in cellular
Cbl utilization and metabolism.
Prenatal detection of fetuses with defects in the complementation groups cblA, cblB, cblC, and cblF has been
accomplished using cultured amniotic cells and chemical
determinations on amniotic ¯uid or maternal urine. In
several cases, in utero Cbl therapy was done with apparent
success.
FOLATE
The chemistry, biochemistry, and physiology of folic acid and its
derivatives have been extensively reviewed in earlier editions of
this book,1,2 as well as in several excellent monographs.3,4 A
detailed review by Erbe gives a case-by-case analysis in tabular
form of each patient who had been reported up to 1986 with
veri®ed methylenetetrahydrofolate reductase de®ciency or glutamate formiminotransferase de®ciency.5 Other reviews are also
available.6± 16
The pteridine compounds referred to as ``folates'' participate as
coenzymes in a number of critical 1-carbon transfer reactions,
including those involved in the biosynthesis of purines, pyrimidines (dTMP), serine, and methionine, and in the degradation of
histidine. In the 1930s, at about the same time that pteridine
pigments of butter¯y wings were being isolated and characterized,
Wills and her colleagues determined that the absence of folate
from the diet resulted in a macrocytic megaloblastic anemia.17,18
The structural determination and synthesis of the parent compound
were accomplished in the subsequent decade.19 ``Folic acid'' and
``folate'' are the preferred synonyms for pteroylglutamic acid
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
``leucovorin,'' or ``citrovorum factor,'' is a reduced folate that has
been used therapeutically because of its chemical stability.
Folate Transport
Two distinct systems have been described for the transport of
folates and folate antagonists (antifolates) across mammalian cell
membranes.20,21 One, the reduced folate carrier (RFC), encoded
on chromosome 21q22.2-22.3 ([SLC19A1], NM_003056, MIM
600424), has been studied mostly in cancer cells and mediates a
low af®nity, high-capacity system for the uptake of reduced folates
and methotrexate at high (mM) concentrations.21±25 It shows
considerable transcript heterogeneity;25,26 the putative intestinal
folate transporter has an identical cDNA.27 The second system, a
family of membrane-associated folate-binding proteins (FBP) or
folate receptors (FR), is coded for by genes on chromosome 11
(q13.3-13.5).21,28 These glycoproteins mediate a high af®nity, lowcapacity system and operate at low (nM) concentrations of
exogenous folate. The FR-a ([FOLR1], NM_000802, MIM
136430) and FR-b ([FOLR2], NM_000803, MIM 136425) genes
have similar structures, but differ in their 50 -untranslated regions
and in their transcription regulatory elements. Both FR-a and FR-b
are attached to the cell membrane by a glycosylphosphatidylinositol anchor, and there is evidence for receptor-mediated
internalization (potocytosis).29± 31 The role of nonclathrin-coated
invaginations in the plasma membrane (caveola), the process of
``potocytosis,'' and the linkage of FR and RFC in this process
remain debated.21,32,33 In addition to the above systems, there is
evidence that passive diffusion may work together with folate
receptors in transplacental folate transport.34
Folate Polyglutamates
Fig. 155-1 Structure of folic acid and its derivatives. (Modi®ed from
Rowe.1 Used with permission.)
(PteGlu) and pteroylglutamate, respectively (Fig. 155-1). The term
folate is also used in the generic sense to designate a member of
the family of pteroylglutamates, each having a different level of
reduction of the pteridine ring, 1-carbon substitution, and number
of glutamate residues. In the folate compounds, pteroic acid is
conjugated with one or more molecules of L-glutamate, each
linked by amide bonds to the preceding molecule of glutamate
through the g-carboxyl group. The terms pteroylpolyglutamate and
folate polyglutamate apply to folate compounds with more than
one glutamate residue. The biologically active folates are
substituted derivatives of 5,6,7,8-tetrahydrofolic acid (H4Folate)
(Fig. 155-2).
As summarized in Fig. 155-1, there are at least three stages of
reduction of the pyrazine ring of the pteridine moiety; at least six
different 1-carbon groups substituted at positions N 5, N 10, or both;
and g-glutamyl peptide chains of varying length. 5-MethylH4Folate is the predominant form of folate in serum and in
many tissues. 5-Formyl-H4Folate, also known as ``folinic acid,''
Human cells need a critical concentration of intracellular folate to
allow activity of folate-dependent enzymes. The amount required
to maintain an optimal rate of growth in culture varies from about
50 nM in human ®broblasts to about 1 mM in human lymphocytes
and certain tumor cells.35 Although the Kms for monoglutamate
folates of many of the folate-dependent enzymes are greater than
1 mM, those for polyglutamate folates of appropriate chain length
are generally much lower, allowing folate metabolism to progress
at the concentration of folates present in cells. Both a cytoplasmic
and a mitochondrial folylpolyglutamate synthase add glutamate
residues to selected folate molecules. A single gene on chromosome 9cen-q34 ([FPGS], NM_004957, MIM 136510) with
alternative splice sites codes for the two folylpolyglutamate
synthase proteins.36± 38 These enzymes form a peptide bond
between the g-carboxyl of the glutamate already present and the
a-amino group of the glutamate to be added. Folylpolyglutamate
synthase adds glutamate residues one at a time, requires ATP for
its reaction, utilizes H4Folate and other folates as well as
antifolates as substrates with different af®nities, and reacts poorly
with folic acid and 5-methyl-H4Folate. There is a unidirectional
¯ow of triglutamate forms from the mitochondria to the cytoplasm,
but longer forms cannot exit the mitochondria. Speci®c instances
of channeling of polyglutamate intermediates between active sites
of multifunctional proteins have been demonstrated. Thus,
they may play a role in maintaining speci®c protein-protein
Fig. 155-2 Structure of 5,6,7,8-tetrahydrofolic acid (THF). (Reprouced from Rowe.1 Used with permission.)
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3900
PART 17 / VITAMINS
interactions.39 Cell lines defective in folate polyglutamate
formation have been reported. A mutant Chinese hamster cell
line is auxotrophic for glycine, adenosine, and thymidine,
apparently because reactions generating these within the cell
require folate polyglutamates.40,41 A human breast carcinoma cell
line is defective in the synthesis of methotrexate polyglutamates
and, consequently, is resistant to methotrexate.
Folate polyglutamates must be hydrolyzed in the intestine prior
to absorption, and monoglutamates are released into the circulation.42,43 The g-glutamyl chain is resistant to digestion by the
common proteolytic enzymes and is hydrolyzed by speci®c
pteroylpolyglutamate hydrolase (conjugase) enzymes. Two distinct forms of human conjugase have been described, one in the
intestinal brush border, which acts at neutral pH, and another
within lysosomes. The lysosomal enzyme may play a role in
regulating intracellular polyglutamate levels. Both human ([GGH],
NM_003878, MIM 601509) and rat lysosomal conjugases have
been cloned.44,45 Prostate-speci®c membrane antigen (PSMA) has
conjugase activity.46
The major metabolic pathways of the folates are shown in Fig.
155-3. In most cells, because serine and glycine are the major
sources of 1-carbon units, entry into the active 1-carbon pool of
intermediates is by way of 5,10-methylene-H4Folate. This
compound is used unchanged for the synthesis of thymidylate
(Fig. 155-3, reaction 4). 5,10-Methylene-H4Folate is reduced to
5-methyl-H4Folate for the biosynthesis of methionine (Fig. 155-3,
reaction 1), or is oxidized to 10-formyl-H4Folate for use in purine
synthesis47 (Fig. 155-3, reactions 6 and 7). All the interconversions
of folates involve exchange of side chains between tetrahydrofolates, except for the formation of thymidylate by thymidylate
synthase (Fig. 155-3, reaction 4), which results in the oxidation of
the folate moiety to dihydrofolate (H2Folate).
Folic acid, a synthetic vitamin not found in nature, and
H2Folate are reduced by dihydrofolate reductase (Fig. 155-3,
reaction 5) to H4Folate. Dihydrofolate reductase has long been
known to be the primary site of action of the chemotherapeutic
drug, methotrexate, an antifolate. Unstable gene ampli®cation
resulting in resistance to methotrexate is associated with doubleminute chromosomes, while in stably ampli®ed cells that are
resistant to methotrexate, the ampli®ed genes are associated with
elongated chromosomes.48 The gene for dihydrofolate reductase
has been assigned to chromosome 5q11.1-q13.2 ([DHFR],
NM_000791, MIM 126060).49,50
The major source of single-carbon units in most organisms is
carbon 3 of serine, which is derived from glycolytic intermediates.
Serine hydroxymethyltransferase catalyzes the cleavage of serine
to glycine and 5,10-methylene-H4Folate (Fig. 155-3, reaction 3).
In mitochondria, glycine is also metabolized to 5,10-methyleneH4Folate, plus carbon dioxide and ammonia, by the glycine
cleavage system16 (also see Chap. 90). There are two separate
serine hydroxymethyltransferases, a cytoplasmic form ([SHMT1],
cSHMT, NM_004169, MIM 182144) and a mitochondrial form
([SHMT2], mSHMT, NM_005412, MIM 138450). Both have been
cloned: cSHMT is on chromosome 17p11.2 (NM_004169), and
mSHMT is on chromosome 12q13 (NM_005412).51± 53 The
cytosolic form has been crystallized, and its structure solved.54
A mutant Chinese hamster ovary cell line de®cient in the
mitochondrial serine hydroxymethyltransferase is auxotrophic
for glycine,55 indicating that the cytoplasmic enzyme cannot
take over all the functions of the mitochondrial enzyme. By
catalyzing the conversion of glycine in the diet to serine, which
then can form pyruvate, cSHMT may also play a role in
gluconeogenesis.16 Both forms of serine hydroxymethyltransferase are capable of catalyzing the hydrolysis of 5,10-methenylH4Folate to 5-formyl-H4Folate.56
Two enzyme systems carry out folate interconversions in
mammals.57 A trifunctional polypeptide bears activities of NADPdependent methylene-H4Folate dehydrogenase, methenylH4Folate cyclohydrolase, and 10-formyl-H4Folate synthase (Fig.
155-3, reactions 6, 7, and 8; [MTHFD], NM_005956).58 The
interconversion of 5,10-methylene-H4Folate, 5,10-methenylH4Folate, and 10-formyl-H4Folate links the major source of
Fig. 155-3 Scheme of folate-mediated 1-carbon transfer reactions: 1.
Methionine synthase (methyl-H4Folate:homocysteine methyltransferase); 2. Methylene-H4PtGlu reductase; 3. Serine hydroxymethyltransferase; 4. Thymidylate synthase; 5. Dihydrofolate reductase; 6.
Methylene-H4Folate dehydrogenase (NAD and NADP-dependent
forms have been described); 7. Methenyl-H4Folate cyclohydrolase;
8. 10-Formyl-H4Folate synthase; 9. GAR (5-phosphoribosylglycineamide) transformylase; 10. AICAR (5-phosphoribosyl-5-aminoimidazole-4-carboxamide)transformylase; 11. Glutamate formiminotransferase; 12. Formimino-H4Folate cycloderaminase; 13. 5,10-MethenylH4Folate synthetase; 14. 10-Formyl-H4Folate dehydrogenase; 15.
Glycine cleavage pathway.
Metabolic Pathways and Enzymes
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
single-carbon units, as methylene-H4Folate, with synthesis of
thymidylate (thymidylate synthase [Fig. 155-3, reaction 4]) or
purine (GAR and AICAR transformylase [Fig. 155-3, reactions 9
and 10]). The trifunctional polypeptide also permits either the
release of single carbons from folate as formate or, more probably,
the scavenging of potentially toxic formate (Fig. 155-3, reaction
8). The trifunctional enzyme is found only in the cytosol,59 and is
encoded by a gene on chromosome 14q24.60 A separate
bifunctional NAD-dependent methylene-H4Folate dehydrogenase-cyclohydrolase, without synthase activity, also exists
([MTHFD2], NM_006636). This bifunctional enzyme is not
detected in normal adult tissue but has been found to be expressed
in tissues which contain undifferentiated cells and in transformed
mammalian cells.57,61 It is encoded by a nuclear gene but is found
predominantly in the mitochondria of transformed cells.59 The
crystal structure of the dehydrogenase/cyclohydrolase domain of
the human trifunctional enzyme has been determined in the
presence of NADP,62 as has that of the bifunctional bacterial
enzyme.63 While the NADP binding site is clear, the folate binding
site(s) are only predicted by modeling, and the nature of the
cyclohydrolase active site is not apparent.62
10-Formyl-H4Folate dehydrogenase ([Fig. 155-3, reaction 14)];
[10-FTHFDH], AF052732) releases excess active single-carbon
fragments from the folate pool and generates carbon dioxide. Its
activity is restricted to the liver64 and serves to maintain suf®cient
H4Folate to permit acceptance of single carbons in folatedependent reactions.
5,10-Methenyl-H4Folate synthase is an ATP-dependent
enzyme ([Fig. 155-3, reaction 13]; [MTHFS], NM_006441)65± 67
which converts 5-formyl-H4Folate (folinic acid) to 5,10-methenylH4Folate. Thus, this enzyme is important in supporting the clinical
use of folinic acid for preventing methotrexate toxicity.
5,10-Methylene-H4Folate reductase ([Fig. 155-3, reaction 2];
[MTHFR], AJ237672, MIM 236250)68±70 converts 5,10-methylene-H4Folate to 5-methyl-H4Folate and probably uses only
polyglutamates as substrates within the cell. The human enzyme
binds FAD, uses NADPH as electron donor, and functions as a
dimer of 77 kDa subunits.71,72 It is inhibited by adenosylmethionine, which is bound by the C-terminal regulatory region.72 The
reaction is bidirectional in vitro, but in vivo, it is essentially
unidirectional toward 5-methyl-H4Folate. It is usually assayed in
the reverse direction in vitro, using menadione as electron
acceptor, but it can be assayed in the physiological direction as
well.68 Under the latter conditions, the concentration of adenosylmethionine required for inhibition is considerably smaller than
that required for inhibition of the reverse reaction.69 The human
gene has been cloned and localized to chromosome 1p36.3
(AJ237672) and consists of 11 exons.73,74 The homologous
enzyme from E. coli is considerably smaller (33 kDa) by virtue
of having no adenosylmethionine-binding regulatory domain. Its
crystal structure has been solved.75
Methionine synthase, also known as 5-methyl-H4Folate:Lhomocysteine methyltransferase ([MTR], NM_000254, MIM
156570), is a cobalamin-dependent enzyme that catalyzes the
transfer of a methyl group from methyl-H4Folate (or adenosylmethionine) to homocysteine to form methionine (Fig. 155-3,
reaction 1). In the complete reaction, the methyl group from
methyl-H4Folate is transferred to enzyme-bound cob(I)alamin to
form methylcobalamin. The methyl group is then transferred to
homocysteine, producing methionine and regenerating cob(I)alamin. After a number of cycles, the enzyme-bound cob(I)alamin
oxidizes spontaneously to inactive, enzyme-bound cob(II)alamin,
and a reducing system and adenosylmethionine are required to
reform methylcobalamin and reactivate the enzyme.76±83
Mammalian methionine synthase is an 85-kDa cytoplasmic
enzyme that functions as a monomer. Using the binding of
cobalamin to methionine synthase in extracts of human-hamster
hybrid cell lines as a marker, methionine synthase was assigned to
human chromosome 1.84 The cloning of the gene for human
methionine synthase has con®rmed this assignment at 1q43.85± 88
The predicted sequence of the human enzyme is 55 percent
identical to the cobalamin-dependent methionine synthase from E.
coli86 (bacteria also have a noncobalamin-requiring methionine
synthase). The bacterial enzyme has been extensively studied, and
the structures of its cobalamin-binding and adenosylmethioninebinding domains have been determined by x-ray crystallography.89,90 The structure of the cobalamin-binding domain is
homologous to that of the C-terminal cobalamin-binding domain
of methylmalonyl CoA mutase, the other cobalamin-requiring
mammalian enzyme (see Chap. 94).
Because the circulating form of folate in humans is methylH4Folate monoglutamate and because the methylene-H4Folate
reductase reaction is essentially irreversible in the cell, folate
entering cells must pass through the methionine synthase reaction
in order to generate tetrahydrofolate and the other folate
cofactors.91±95 In cobalamin de®ciency (acquired or inherited,
see below), or when cobalamin is irreversibly oxidized by nitrous
oxide,96 methionine synthase activity decreases or is absent,
methyl-H4Folate and homocysteine accumulate, and methionine
and, especially, adenosylmethionine are reduced. In addition to the
folate being ``trapped'' as methyl-H4Folate, most of it remains as
the monoglutamate because methyl-H4Folate is a poor substrate
for the folylpolyglutamate synthase enzyme. Folic acid or folinic
acid can bypass this block until methyl-H4Folate again accumulates as a result of methylene-H4Folate reductase activity.
In E. coli, the reductive activation system that maintains the
active form of the cobalamin cofactor on methionine synthase is a
two-component ¯avoprotein system97,98 consisting of ¯avodoxin99
and ¯avodoxin reductase.100 A similar system had been postulated
for eukaryotes. Recently, based on the hypothesis that the
mammalian enzyme would be a multifunctional protein incorporating both reductase activities, Gravel and colleagues cloned an
enzyme called methionine synthase reductase (MSR), which is at
least one component of this system.81,83 MSR is a unique member
of the ferredoxin-NADP‡ reductase family of electron transferases, combining binding sites for FMN and FAD, along with
NADPH. The biochemical details of the reactivation reaction are
unknown, and it remains unclear whether another protein may be
involved. MSR has a predicted molecular size of 77 kDa and is
encoded by a gene on chromosome 5p15.2-15.3 ([MTRR],
NM_002454, MIM 602568).83
During the catabolism of histidine, a formimino group is
transferred to H4Folate, which transfer is followed by the release
of ammonia and generation of 5,10-methenyl-H4Folate. The two
enzyme activities, glutamate formiminotransferase (Fig. 155-3,
reaction 11) and formimino-H4Folate cyclodeaminase (Fig. 155-3,
reaction 12), share a single polypeptide which channels folate
polyglutamate molecules from one reaction to the next.101,102 The
pathway represents only a minor source of single-carbon folates
and may exist only in liver and kidney; the enzymes seem to be
absent from ®broblasts and blood cells.
Disorders of Folate Nutrition,
Transport, and Metabolism
Nutritional Disorders. Although a number of children born to
mothers with a diet de®cient in cobalamin have shown evidence of
cobalamin de®ciency (see ``Cobalamin (Vitamin B12)'' below),
folate de®ciency in the infant secondary to de®ciency in the
mother is unusual.15 In nutritional folate de®ciency in adults, as
described in Herbert's classic self-study,103 the peripheral blood
and bone marrow changes that occurred after 4 months were
preceded by a much-earlier fall in serum folate and a rise in
urinary FIGLU levels. Psychologic and mental changes followed,
but were rapidly reversed by folic acid supplementation. Red
blood cell folate levels fall in folate de®ciency signi®cantly later
than do serum folate levels. On the other hand, there are some
situations in which there are no defects in folate metabolism per
se, but in which folate therapy has been suggested. These include
supplements of folic acid given to pregnant women to produce an
increase in the mean birth weight of infants104 and, particularly,
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3902
PART 17 / VITAMINS
Fig. 155-4 Processes and reactions affected by inherited disorders
of folate transport and metabolism; 1. Methylene-H4Folate reductase
de®ciency; 2. and 3. Functional methionine synthase de®ciency
(cblE, methionine synthase reductase de®ciency; cblG, methionine
synthase de®ciency); see text; 4. Glutamate formiminotransferase
de®ciency; 5. Hereditary folate malabsorption Ð A, dihydrofolate
reductase de®ciency; B, methenyl-H4Folate cyclohydrolase de®ciency; C, cellular uptake defect of folate; and D, methyl-H4Folate:homocysteine methyltransferase de®ciency (original report from
Japan9). Disorders involving folate transport are indicated by a
broken line, whereas those involved in folate metabolism are
indicated by a solid line. The numbered steps show the sites of
well-characterized inherited disorders of folate transport or metabolism. Steps are the diseases that have been presented in the
literature; those that remain in dispute are indicated with letters.
AdoMet 5 adenosylmethionine; H2folate 5 dihydrofolate; H4Folate
5 tetrahydrofolate; methyl-B12 5 methylcobalamin; GAR 5 5-phosphoribosylglycinamide; FGAR 5 a-N-Formyl-glycinamide ribonucleotide; AICAR 5 5-phosphoribosyl-5-aminoimadazole-4-carboxamide; C2, C8 5 carbons number 2 and 8 or of purine ring.
supplements given prior to conception to women at risk for bearing
a child with neural tube defects to reduce the frequency of these
disorders.105 It has also been suggested that increased folate intake
may serve to reduce serum homocysteine concentration, a likely
risk factor in peripheral vascular disease (see Chap. 88).106±108
The processes and reactions affected by inherited disorders of
folate transport and metabolism are shown in Fig. 155-4. Those
that are discussed in some detail below include hereditary folate
malabsorption (reaction 5); glutamate formiminotransferase de®ciency (reaction 4); methylene-H4Folate reductase de®ciency
(reaction 1); and functional methionine synthase de®ciency
(reactions 2 and 3).
with severe bilateral pneumonia. He was one of seven siblings, two
of whom had died in the ®rst year of life without de®nitive
diagnosis. In contrast to other cases, there was no sign of mental
retardation, and correction of the serum folate levels did result in
correction of the levels of folate in the cerebrospinal ¯uid
(CSF). There is evidence for parental consanguinity in four
families.112,114,116,121
The common clinical presentation in hereditary folate malabsorption is megaloblastic anemia in the ®rst few months of life
with low serum folates. Laboratory ®ndings may include urinary
excretion of formiminoglutamic acid (FIGLU) and orotic
acid.12,120 All patients were severely restricted in their ability
to absorb oral folic acid or oral reduced folates. Large doses of
oral folates did cause a hematologic response in some
patients.111,112,120 Parenteral therapy with folates has been
effective in correcting anemia, but has been of limited effectiveness in correcting the levels of folate in the CSF. Other studies
have suggested that folinic acid118,119 or methyl-H4Folate is more
effective in increasing CSF. There is signi®cant clinical heterogeneity among patients. In some patients, seizures were ameliorated by folate therapy, while in others, they were exacerbated by
it. It has been noted125 that the presence of seizures, with or
without cerebral calci®cations, is coincident with the ability to
respond hematologically to large doses of oral folinic or folic acid;
the reason for this is not known.
One of the patients120 had additional ®ndings, including a
relative inability to retain plasma folate after parenteral folate
administration, a ®nding also seen in another patient;114 high
levels of folate in the red blood cells following folate therapy; low
normal plasma levels of methionine; the presence of cystathionine
Hereditary Folate Malabsorption (MIM 229050)
Clinical and Laboratory Findings. This disorder [Fig. 155-4 (5)],
which has also been called congenital malabsorption of folate
because of its early clinical presentation, has been described in
fewer than 20 patients, mostly females.109±123 The disease is
characterized by severe megaloblastic anemia. Diarrhea, mouth
ulcers, and failure to thrive are common, and most patients showed
progressive neurologic deterioration. Folinic acid-responsive
peripheral neuropathy has been described.115,123 Among the
patients were two pairs of sisters,111,124 and there may have
been additional unrecognized affected patients in these families,
because one patient had a sib who died at age 3 months119 whose
sex was not reported. Another patient, who was one of nine
children, had sisters who died shortly after birth; in addition, she
had a brother who died at the age of 13 years, but no further
clinical details were provided.120 A report from Israel121 described
a boy with this disorder, an infant who presented at age 4 months
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
in the CSF and a response of the patient to methionine therapy; and
increased susceptibility to infections associated with low levels of
serum IgM and IgA. One of the affected boys121 had a partial
de®ciency in both humoral (surface Ig and response to pokeweed
mitogen) and cellular (E-rosette forming and response to
hemagglutinin and concanavalin A) immunity.
These patients with hereditary folate malabsorption provide the
best evidence for the existence of a speci®c carrier for folate both
at the level of the intestine and the choroid plexus. Oxidized and
reduced folates must share this system, because the absorption of
both is effectively blocked in these patients. The same gene
product must mediate both intestinal transport and transport of
folates into the brain because, except in the two affected
males,121,123 levels in the CSF remained low when blood folate
levels were raised suf®ciently to correct the anemia. As mentioned
earlier, a cDNA for the putative intestinal transporter has been
cloned, and it is identical to that for the reduced folate carrier.27 It
is likely that uptake of folates into other cells of the body is normal
in patients with hereditary folate malabsorption, because a
hematologic response occurs in the presence of relatively low
blood folate levels. In addition, the content and distribution of
folates were normal in cultured ®broblasts from the one patient
studied.120 Thus, it will be interesting whether mutations in the
putative gene for intestinal transport will be found in patients with
hereditary folate malabsorption.
Treatment. Cooper has stressed15 that it is essential to maintain
folate levels in the serum, red blood cells, and CSF above levels
associated with folate de®ciency (4, 150, and 15 ng/ml,
respectively). As mentioned above, some patients may respond
to large oral doses of folic acid, folinic acid, or methyltetrahydrofolic acid. Oral doses may be increased to 100 mg/day or more
if necessary.15 If oral therapy does not work, systemic therapy
must be instituted with daily injections (subcutaneous, intramuscular, or intravenous) of folinic acid.126 If CSF folate levels cannot
be normalized, periodic intrathecal injections should be considered.15
Genetics. The occurrence of at least one sibship with hereditary
folate malabsorption and the documented cases of consanguinity
suggest inheritance as an autosomal recessive disorder. All but
four of the documented cases121,123,126 have been female, although
in one of the families, there is the suggestion of another possibly
affected male.120 In the father of one patient, the absorption of oral
folate was seen to be intermediate,114 again suggestive of
autosomal recessive inheritance.
Cellular Uptake Defects. These disorders (Fig. 155-4 (C))
appear in a group of reported patients with varied clinical ®ndings,
some of which were associated with serious hematologic disease.
Although the individual abnormalities of folate uptake are well
characterized, it remains unclear whether these disorders represent
primary inherited abnormalities.
Branda et al. reported a patient with severe aplastic anemia that
responded to high doses of folate therapy.127 The patient was part
of a large kindred in which there was a high incidence of severe
hematologic disease, including anemia, pancytopenia, and leukemia. These diseases were found in 34 individuals in four
generations, resulting in the death of 18. The proband showed a
marked reduction of the uptake of methyl-H4Folate in stimulated
lymphocytes despite a normal uptake of folic acid. Among eight
healthy family members, including three of the proband's children,
four were found to have a similar abnormality. In addition, there
was a less marked reduction in the uptake of methyl-H4Folate by
bone marrow cells from the proband and his son. Of particular
interest, however, was the ®nding that one son showed initially
normal folate uptake, but neutropenia subsequently developed, and
then the abnormality was exhibited. This observation has been
taken to suggest that this disorder may not be a primary defect in
folate uptake.125 Folate uptake by erythrocytes and the intestinal
absorption of folate were found to be normal. Since the original
report, the patient died at age 41 due to respiratory failure
secondary to pleural effusion and ascites.128 Three children in the
family had an increased incidence of sister chromatid exchange.
An additional family was described with a transport defect
which affected red cells and bone marrow, but not lymphocytes.129
The proband and his daughter had dyserythropoiesis without
anemia; three brothers were normal. Erythrocytes from the patient
showed abnormalities in the Vmax and total uptake of methylH4Folate, whereas folic acid uptake was normal; the daughter
showed only a possible elevation in the Km for methyl-H4Folate,
while the three clinically normal brothers resembled the proband
kinetically. The status of both of these disorders of cellular uptake
remains to be clari®ed.
An 18-year-old male with progressive neurologic disease,
which included sensorineural hearing loss, a cerebellar syndrome,
distal spinal muscular atrophy, and pyramidal tract dysfunction,
had an isolated folate de®ciency in the CSF and normal serum and
red blood cell folate levels.126,130,131 The defect may lie in the
isolated transport of folate into the CSF and may turn out to be a
variant of hereditary folate malabsorption.
Dihydrofolate Reductase De®ciency Ð Suspect Disorder. There
are two published reports describing three cases of putative
dihydrofolate reductase de®ciency132,133 [Fig. 155-4 (A)]. Megaloblastic anemia developed in these patients soon after birth and
showed a better clinical response to folinic acid (5-formylH4Folate), a reduced folate, than to folic acid, an oxidized folate.
In all three patients, dihydrofolate reductase activity was
decreased in liver biopsies.
The original patient132 had a reduction in dihydrofolate
reductase activity in the liver to 35 percent of control values
(more than 2 SD lower than autopsy liver samples in seven control
subjects). This male had anemia at 6 weeks of age, which
subsequently became megaloblastic. Oral doses of 50 to 500 mg/
day of folic acid did not produce a clinical response; 5 mg/day of
oral folic acid resulted in a sustained 3-year remission. When
folate therapy was discontinued, the patient relapsed. Small doses
of folinic acid were effective in producing a remission. At age 19
years,5 he was not grossly mentally retarded but had manifested
``sociopathic and frankly criminal behavior that resulted in
repeated incarcerations.''5 Although he was still folate-dependent,
extracts of cultured ®broblasts showed normal total activity,
kinetics, and heat stability of dihydrofolate reductase.
Two unrelated patients were later reported with neonatal
megaloblastic anemia that was attributed to dihydrofolate
reductase de®ciency.133 Activity in a liver biopsy was not
detectable in the routine assay in the ®rst case, but normal levels
(1.0 to 1.7 nM of dihydrofolate reduced per min per mg of protein)
were found in the presence of 0.6 M potassium chloride. At age 3
years, her bone marrow showed dihydrofolate reductase activity
that was 10 percent of control levels and a heat-labile enzyme with
a molecular size of 58,000 daltons, considerably higher than that
of the normal enzyme.134 At age 9 years,1 the child was severely
mentally retarded and still showed folate-dependent macrocytic
anemia. We have shown that the correct diagnosis in this child is
methionine synthase reductase de®ciency (cblE complementation
group, see below).
The second child was ®rst seen at age 26 days because of oral
and anal moniliasis and poor feeding. Low neutrophil and platelet
counts were seen, and over the next 2 weeks a megaloblastic
anemia developed. The serum folate level at 9.5 ng/ml was
borderline normal for his age,1 and the serum cobalamin level was
normal. Dihydrofolate reductase activity in a liver biopsy specimen was 20 percent of the normal median value and was activated
about twofold by 0.6 M potassium chloride, similar to the control
liver samples. Subsequent study revealed that the patient was
de®cient in functional transcobalamin II135 (see cobalamin section
below). There was absent unsaturated serum cobalamin-binding
capacity, although immunoassay did show transcobalamin II
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3904
PART 17 / VITAMINS
protein levels at 39 percent of the normal mean. There was no
cobalamin-binding protein corresponding to transcobalamin II on
Sephadex-gel chromatography. The patient was reinvestigated
because of the development of mental retardation and severe
neuropathy after 2 years of treatment.136 It was concluded that this
patient had functionally inactive transcobalamin II of the type
described by Seligman.137
No additional patients have been described. Although at least
two of the reported children had inborn errors of cobalamin
metabolism, which were not initially recognized, the low liver
values of dihydrofolate reductase remain dif®cult to explain. Of
interest, urinary amino acids were reported to show a normal
pattern, and no FIGLU was detected in the urine of the two
patients who were reported in the most detail.133 Thus, although
the possibility of dihydrofolate reductase de®ciency in an infant
with severe megaloblastic anemia must be considered, all other
known causes must be ruled out before this diagnosis can be
con®rmed.
Methenyltetrahydrofolate Cyclohydrolase De®ciency Ð Suspect Disorder. As previously discussed, methenyl-H4Folate
cyclohydrolase (Fig. 155-3, reaction 7) is part of a trifunctional
protein that contains the activities of methylene-H4Folate
dehydrogenase, methenyl-H4Folate cyclohydrolase, and 10-formyl-H4Folate synthase.138,139 Methenyl-H4Folate cyclohydrolase
de®ciency [Fig. 155-4 (B)] was proposed in three children who
had 44 percent of control enzyme activity on liver biopsy and
levels of 58 percent, 36 percent, and 43 percent of control values in
erythrocytes.127 Clinically, the patients had mental retardation,
microcephaly, ventricular dilatation, and abnormal electroencephalograms. A later report from the same laboratory9 essentially
retracted the diagnosis, and no additional cases have been
reported.
Glutamate Formiminotransferase De®ciency (MIM 229100).
As a result of the catabolism of histidine, a formimino group is
transferred to tetrahydrofolate, followed by the release of
ammonia and the formation of 5,10-methenyl-H4Folate. The two
enzyme activities involved in these steps, glutamate formiminotransferase (EC 2.1.2.5) (Fig. 155-3, reaction 11) and formiminoH4Folate cyclodeaminase (EC 4.3.1.4) (Fig. 155-3, reaction 12),
share a single polypeptide, which forms an octameric
enzyme101,102 that channels polyglutamate folates from one
reaction to the next. This pathway represents a minor source of
single-carbon units and may be present only in liver and kidney.
Clinical and Laboratory Presentation. Reports on fewer than 20
patients have been published, and it is not clear whether this
enzyme de®ciency is associated with a disease state or whether the
association of clinical ®ndings with FIGLU excretion is a result of
bias of ascertainment.5,126,131,140 Individuals with glutamate
formiminotransferase de®ciency [Fig. 155-4 (4)] have been
described with two distinct phenotypes. In one type, there is
mental and physical retardation, cortical atrophy with dilatation of
cerebral ventricles, and abnormal electroencephalograms. The
second type shows no mental retardation but massive excretion of
FIGLU. It has been postulated that the severe form is associated
with a major block in the cyclodeaminase activity and the mild
form with a block in the formiminotransferase activity,1 but no
direct enzyme measurements have been presented to support this
hypothesis.
Diagnosis of these diseases is hampered by the absence of
enzyme activity from cultured human cells,12 and there is dispute
as to whether the de®ciency can be diagnosed using red blood
cells.5,141 Indeed, in most cases in which the liver has been
examined, enzyme activities were higher than would have been
expected for a complete block resulting in disease.9 Erbe5 has
summarized and tabulated most of the known patients with
glutamate formiminotransferase de®ciency.124,142±155 The patients
have come to medical attention from 3 months to 42 years of age.
Three patients presented with delayed speech, two had mental
retardation, and two presented with seizures. Two were studied
because they were sibs of known cases. Mental retardation was
described in most of the original Japanese patients,9 whereas only
three of the eight remaining patients were reported to show
evidence of mental retardation.149,151,152,154 Abnormal electroencephalograms and hypotonia have been described frequently.
Several patients showed hematologic ®ndings, including hypersegmentation of neutrophils and macrocytosis. The reported
biochemical ®ndings include: increased urinary, as well as serum,
FIGLU, especially after a histidine load; normal to high serum
folate levels with normal cobalamin levels; hyperhistidinemia;
hypomethioninemia; and histidinuria.
In several of the Japanese patients, FIGLU excretion was
elevated only after histidine loading. Amino acid levels in plasma,
including histidine, were usually normal, but occasionally low
methionine levels were seen.124,154 Urinary excretion of 4-amino5-imidazolecarboxamide,149,156 an intermediate metabolite in
purine synthesis, has been reported, as has excretion of hydantoin-5-propionate, the stable oxidation product of the FIGLU
precursor, 4-imidazolone-5-propionate.15,154,155
Three patients of 12 months, 3.3 years, and 5.5 years, with a
neuroblastoma, a germ cell tumor, and a ®bromatous sarcoma,
respectively, were found to have increased excretion of FIGLU and
hydantoin propionic acid.140 High levels persisted after treatment,
and it was concluded that the patients had glutamate formiminotransferase de®ciency.
Enzyme Activity. Enzyme activity was measured in the livers of
®ve patients and ranged from 14 to 54 percent of the activity in
control livers; what these values signify is not yet known. In three
families, the level of enzyme activity was said to be low in
erythrocytes; on the other hand, several laboratories have
been unable to detect enzyme activity in erythrocytes, even in
controls.5
Treatment. Response to therapy has been judged on the basis of
decreased urinary excretion of FIGLU. Two patients in one family
responded to treatment with folates;150 six others did not.5 One of
two patients152,154 responded to methionine supplementation.
Given that the correlation between clinical phenotype and FIGLU
excretion remains uncertain, the basis for treating these patients is
unclear.
Genetics. Glutamate formiminotransferase de®ciency has been
found in both male and female offspring of unaffected parents. No
consanguinity has been described. The de®ciency is presumed to
be inherited as an autosomal recessive. In the absence of
detectable enzyme activity in cultured cells, de®nitive resolution
of the inheritance of this disorder awaits the cloning of the human
gene and the localization of the primary defect, because it is likely
that the primary defect could then be detected in DNA from
patients. DNA from putative patients should be put aside to await
molecular diagnosis.
Differential Diagnosis. The major dif®culty in the diagnosis of
this disorder lies in the lack of expression of enzyme activity
outside of the liver. Aside from FIGLU excretion in the urine and
assay of enzyme activity in liver biopsy, which in reported cases
has shown unusually high residual activities,9 de®nitive diagnosis
is dif®cult. In addition, FIGLU excretion may be caused by other
defects in folate or cobalamin metabolism. Indeed, ®broblasts of
one patient, who had megaloblastic anemia and folate-responsive
homocystinuria,141 were examined further. This patient has low
methionine biosynthesis, low methionine synthase activity, and
low MeCbl, and, indeed, has methionine synthase de®ciency (cblG
complementation group, see below). Thus, it is appropriate to
study ®broblasts from all patients who show evidence of
hypomethioninemia for evidence of a functional de®ciency in
methionine synthase.
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
Methylenetetrahydrofolate Reductase De®ciency (MIM
236250). Methylene-H4Folate reductase (EC 1.5.1.20) is a cytoplasmic enzyme that catalyzes the NADPH-linked reduction of
methylene-H4Folate to methyl-H4Folate (Fig. 155-3, reaction 2).
Methyl-H4Folate serves as the methyl donor for the methylation
of homocysteine in the reaction catalyzed by methionine synthase
(5-methyl-H4Folate:homocysteine methyltransferase [Fig. 155-3,
reaction 1]). The combined action of methylene-H4Folatereductase
and methionine synthase supplies single-carbon units for methylation reactions that use adenosylmethionine. The reaction catalyzed
by methylene-H4Folate reductase is essentially irreversible under
physiological conditions, and enzyme activity is regulated by levels
of adenosylmethionine, which is an inhibitor.69,157,158
Clinical and Laboratory Findings. Since the ®rst reports of
methylene-H4Folate reductase de®ciency in 1972159,160 [see Fig.
155-4 (1)], more than 40 cases have been reported.131,161±197 The
major biochemical ®ndings have been moderate homocystinuria
and hyperhomocystinemia with low or relatively normal levels of
plasma methionine. The clinical severity of this disorder varies
greatly from case to case, with most patients being symptomatic in
infancy or early childhood, but the age of diagnosis has ranged
from before birth to adulthood.159,182,193,198. An infant showed
extreme progressive brain atrophy and demyelinization on MRI.197
A 10-year-old male exhibited a developmental history and
physical signs compatible with Angelman syndrome.199 In a
family with six sibs, three patients had severe recurrent strokes in
their early 20s, resulting in the death of two of them 1 year after
clinical onset.193 Two of these patients were noted to have a
marfanoid habitus, although this is not a frequently reported
®nding. In another family, a younger brother developed limb
weakness, incoordination, paresthesias, and memory lapses at age
15 years and was wheelchair-bound by his early 20s, whereas his
older brother was asymptomatic at age 37 years.194
The most common clinical manifestation in methyleneH4Folate reductase de®ciency is developmental delay. Motor and
gait abnormalities, seizures, and psychiatric manifestations have
been reported.195,200,201 In Erbe's 1986 clinical review,5 about half
of the patients were microcephalic; EEG abnormalities were
present in most; some abnormalities of gait were described in
almost all patients who were old enough to walk. Homocystinuria
was present in all patients, with a reported range of 15 to 667 mM/
24 h and a mean of 130 mM/24 h. Homocystine, not normally
detected in urine or free in plasma, was found in the plasma: mean
value 57 mM (range: 12 to 233 mM). Although data on total
plasma or serum homocysteine (tHcy) are scarce, levels of 60 to
184 mM (controls: 4 to 14 mM) have been reported.194,202±204
Plasma methionine levels were low in all patients, ranging from 0
to 18 mM, with a mean of 12 mM; normal is 23 to 35 mM,5
although values vary among laboratories.
Although homocystinuria was consistently seen in all patients,
and indeed is the clinical clue by which the diagnosis of
methylene-H4Folate reductase de®ciency is made, the excretion
of homocystine in urine is much less than that found in
homocystinuria due to cystathionine synthase de®ciency (see
Chap. 88). Indeed, it may not be detected on spot testing, which,
therefore, should not be used in isolation to diagnose the
disease.205 The methionine levels in methylene-H4Folate reductase de®ciency are always low-normal or low. This, again,
distinguishes these patients from those with cystathionine synthase
de®ciency, who generally have hypermethioninemia. In contrast to
patients who are functionally de®cient in methionine biosynthesis
because of abnormalities in methylcobalamin formation (complementation groups cblC, cblD, cblE, cblF, and cblG; see below),
patients with methylene-H4Folate reductase de®ciency do not have
megaloblastic anemia. In addition, in contrast to patients with the
cblC, cblD, and cblF disorders, these patients have no methylmalonic aciduria. Although serum folate levels were not always low,
many of the patients with methylene-H4Folate reductase de®ciency had serum folate levels that were low on at least one
determination. In contrast, serum cobalamin levels were almost
always normal. Although the levels of neurotransmitters in the
cerebrospinal ®eld have been measured in only a minority of
patients, they have usually been low.5,200
Studies on Cultured Cells. A de®ciency of methylene-H4Folate
reductase has been con®rmed on studies of liver, leukocytes, and
cultured ®broblasts and lymphoblasts. The enzyme assay routinely
used for these studies measures the activity in the nonphysiological direction, using radioactive methyl-H4Folate. Activity is
extremely sensitive to the stage of the culture cycle of ®broblasts,
with the speci®c activity in control cells being highest in con¯uent
cultures.158 This variability is suf®ciently great to allow for the
misclassi®cation of controls and heterozygotes if not taken into
account. In general, there is rough correlation between residual
enzyme activity and the clinical severity. Both the measurement of
the proportion of folate present in cultured cells as methylH4Folate171 and the synthesis of methionine from labeled
formate178,206 provide a better correlation with clinical severity.
Studies on cultured ®broblasts164,171 and liver177,186 determined
the levels and distribution of folate derivatives. In both control and
mutant ®broblasts, most of the folates present were polyglutamates, and the proportion of polyglutamates relative to folate
monoglutamate was similar; a direct relationship was found in
cultured ®broblasts between the proportion of cellular folate which
was methyl-H4Folate and both the clinical severity and the
residual enzyme activity, indicating that the distribution of the
different folates may be an important control of intracellular folate
metabolism.171
Control cultured ®broblasts can grow when homocysteine,
along with folate and cobalamin, is substituted in the culture
medium for methionine, an essential amino acid for these cells. In
contrast, ®broblasts from patients with methylene-H4Folate
de®ciency do not grow on homocysteine.160,165 This inability to
grow on homocysteine is shared by ®broblasts from patients who
are functionally de®cient in methionine synthase (cblC, cblD,
cblE, cblF, and cblG; see cobalamin section below).207
A differential microbiologic assay that makes use of the fact
that Lactobacillus casei can utilize methyl-H4Folate for growth but
that Pediococcus cerevisiae cannot is a useful screening test for
methylene-H4Folate de®ciency, as analysis requires only small
numbers of cultured ®broblasts.164
Genetic heterogeneity in the severe form of this disorder was
suggested by the fact that ®broblast extracts from two of the
original families showed differential heat inactivation at 55 C.165
Although several of the later onset patients had a thermolabile
reductase under these conditions, thermolability was also found in
patients with early onset disease.208 In some patients, this was
shown to be due to the presence of severe methylene-H4Folate
reductase mutations in combination with the common 677C ! T
mutation that is responsible for the majority of enzyme
thermolability in the general population.209,210
Kang and his colleagues originally suggested that thermolability of reductase activity at 46 C for 5 min in lymphocyte
extracts of adults may be associated with ``intermediate homocystinemia'' and an increased risk for vascular disease in adult
life.211±214 Because of the dif®culties in performing the assay for
methylene-H4Folate reductase on small cell numbers, there were
not many studies designed to test this hypothesis. It is now clear
that hyperhomocystinemia is a risk factor for vascular disease.215±219 The ability to test the role of methylene-H4Folate
reductase thermolability as a contributor to the pathology was
greatly aided by the cloning of the gene220 and the discovery that a
common mutation, 677C ! T, which converts an evolutionarily
conserved alanine at amino acid residue 222 to valine (A222V), is
responsible for the thermolability.209 The T allele was found to
have a frequency of 35 to 40 percent in French-Canadians and
other North Americans, but the frequency may vary in different
ethnic groups.72,209,221,222 In particular, it was very low in samples
from Africa and parts of Asia.222 The association between
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3906
PART 17 / VITAMINS
homozygosity for the T allele and plasma homocysteine levels in a
population was found to be related to the folate status of the
population, with elevations of homocysteine being dependent on
the presence of lower plasma folate levels.223± 225. The role of the
677C ! T polymorphism as a risk factor for vascular disease and
for neural tube defects226 remains a subject of great interest and
debate.221,225± 231 Interestingly, the 677C ! T polymorphism was
found to be associated with a decreased risk of colon cancer.232
Another polymorphism, 1298A ! C, which converts glutamate to
alanine at amino acid residue 429 (E429A), is also associated with
decreased enzyme activity.233±235 A silent genetic variant,
1317T ! C in the same exon, is common in Africans and may
interfere with detection of the 1298A ! C polymorphism;235
677C ! T and 1298A ! C have not been found together in doubly
homozygous form.234,235
Pathophysiology. The prominent biochemical manifestations of
methylene-H4Folate reductase de®ciency include: (a) homocystinuria and homocystinemia; (b) hypomethioninemia; (c) decreased proportion of intracellular folate as methyl-H4Folate; and
(d) decreased neurotransmitter levels. Patients with this disease
rarely have megaloblastic anemia, suggesting that there is not a
folate-related defect in purine and pyrimidine biosynthesis. The
relative importance of homocysteine excess and methionine
de®ciency in these patients remains a matter of conjecture.
The neurologic ®ndings in monkeys treated with nitrous
oxide, an agent that inactivates methionine synthase, are
reported to be similar to that caused by cobalamin de®ciency;91±93
this effect is reversed by methionine therapy. Patients
with disorders of cobalamin metabolism,236 who also have a
block in methionine biosynthesis, may have neurologic deterioration, but they also have hematologic abnormalities that are absent
in methylene-H4Folate reductase de®ciency. The pathologic
changes5,163,166,168,184± 186,189,192 in the patients with methyleneH4Folate reductase de®ciency include dilated cerebral ventricles,
internal hydrocephalus, microgyria, and low brain weight. Also
seen in the brain are perivascular changes, demyelination,
macrophage in®ltration, gliosis, and astrocytosis. Other major
pathologic ®ndings are thromboses of arteries and cerebral veins;
these appear to have been major factors in the death of these
patients. These thromboses are the only pathologic ®ndings shared
with cystathionine synthase de®ciency. It has been suggested that
the combination of methylene-H4Folate reductase de®ciency and
Factor V Leiden may contribute to the vascular pathology in some
patients.237,238 One patient with methylene-H4Folate reductase
de®ciency had a ®brosarcoma.192
It has been pointed out192 that the neuropathologic vascular
®ndings in methylene-H4Folate reductase de®ciency are similar to
those seen in classical homocystinuria due to cystathionine
synthase de®ciency. However, in methylene-H4Folate reductase
de®ciency, it is necessary to explain the demyelination, astrogliosis, and lipid-®lled macrophages, which are associated in many
patients with a progressive course of seizures, microcephaly, and
severe psychomotor retardation.
Two reports189,192 have described classical ®ndings of subacute
combined degeneration of the cord similar to that observed in
patients with untreated cobalamin de®ciency in patients dying with
methylene-H4Folate reductase de®ciency. It has been proposed
that methionine de®ciency causes demyelination, presumably by
interfering with methylation.
Methylene-H4Folate reductase is present in mammalian
brain.239,240 Because several authors have suggested that only
methyl-H4Folate among the natural folates can cross the bloodbrain barrier,172,241 methylene-H4Folate reductase de®ciency may
result in functionally low folate levels in the brain. Because
neurologic symptoms may be observed in patients without very
low methionine levels, it has been suggested77 that the neurologic
dysfunction may occur as a result of impaired purine and
pyrimidine synthesis in the brain, as opposed to low levels of
adenosylmethionine.
The relative importance of low folate levels, low methionine
levels, and low levels of neurotransmitters in the pathology of
methylene-H4Folate reductase de®ciency is uncertain.242 Differences seen between functional methionine synthase de®ciency236,243
(cblC, cblD, cblE, cblF, and cblG) and methylene-H4Folate
reductase de®ciency should be useful in sorting out the relative
importance of low levels of reduced folates, other than methyleneH4Folate, and low levels of methionine. These comparisons have the
potential of being made more dif®cult by developmental and tissue
differences in the distribution of these enzyme activities.244,245
The most important ®nding in the clinical differential diagnosis
is the absence of megaloblastic anemia in patients with methyleneH4Folate reductase de®ciency as compared to patients with
functional methionine synthase de®ciency (cblC, cblD, cblE,
cblF, and cblG complementation groups), and the absence of
methylmalonic aciduria as compared to patients with cblC, cblD,
and cblF disease (see below). It has been shown that levels of
methylcobalamin and of methionine synthase may be low in
®broblasts from some patients with methylene-H4Folate reductase
de®ciency and that this could lead to the incorrect diagnosis of
methionine synthase de®ciency (cblE or cblG).208
Treatment. Methylene-H4Folate reductase de®ciency is very
resistant to treatment but betaine has improved the overall
prognosis.5,15,126,131,195 The rationale for therapy includes: (a)
folates, such as folic acid or folinic acid, in an attempt to maximize
any residual enzyme activity; (b) methyl-H4Folate to replace the
missing product; (c) methionine to correct the cellular methionine
de®ciency; (d) pyridoxine to lower homocysteine levels, because
of its role as a cofactor for cystathionine synthase; (e) cobalamin,
because of its role as a cofactor for methionine synthase; (f )
carnitine, because its synthesis requires S-adenosylmethionine; (g)
betaine,176 because it is a substrate for betaine:homocysteine
methyltransferase,245 a liver-speci®c enzyme that converts homocysteine to methionine; and (h) ribo¯avin, because of the ¯avin
requirement of methylene-H4Folate reductase.
Criteria for the success of treatment5 have included reduction
of the plasma homocysteine levels with elevation of plasma
methionine levels to normal, along with improvement in the
clinical picture. In most cases, several of the agents mentioned
above have been used in combination, and it is somewhat dif®cult
to assess the ef®ciency of a single one.
Cooper15 suggested a therapeutic regimen consisting of oral
betaine, folinic acid, and methionine, with additional vitamin B6
and cobalamin. Cooper recommended cobalamin because of the
observations of subacute combined degeneration of the cord189 in
a child treated with methyl-H4Folate alone. Interestingly, therapy
with methionine alone or with methyl-H4Folate is not particularly
effective in most cases, even though adenosylmethionine de®ciency in the central nervous system appears to be playing a major
role in the pathogenesis of this disease.242 Fowler reported that one
patient responded to ribo¯avin.131 Supplementation with pyridoxine also has been suggested in order to enhance the transsulfuration pathway.195
Therapeutic successes include a patient who was treated with a
combination of methionine, oral folinic acid and vitamin B6, and
cobalamin,179,180 and several patients in whom betaine was
included in the regimen.5,181,182,190 One patient who responded
to betaine at doses of 20 g/day had not responded to other
treatments, including folates and methionine. Cobalamin had not
been used in this patient. The two patients who were treated from
the ®rst month of life190 with folic acid and betaine had normal
psychomotor testing at around the age of 5 years. Ronge and
Kjellman described a 7.5-year-old female, with slight microcephaly, impaired vision, and moderate developmental delay, who
was treated from infancy with 3 to 6 g of betaine daily. She
developed an unexplained increase in appetite and weight gain
from age 4 years. With treatment, her previously undetectable
plasma methionine levels normalized, but total plasma homocysteine levels remained elevated.202
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
Thus, betaine181,190,202,246 appears to be the most promising
agent for therapy of methylene-H4Folate reductase de®ciency,
although, as mentioned above, some of the other therapies have
been partially successful. There is not a great deal of data on the
optimum dose of betaine in these patients, but Ronge and
Kjellman suggested a dose of 6 g/day (3gb.i.d.) but indicated
that they intended to increase the dose to 12 g/day in their
patient.202 Ogier de Baulny and colleagues suggested a dose of 2
to 3 g/day in young infants and 6 to 9 g/day in children and
adults.195 Kakura and colleagues studied the relationship of serum
total homocysteine and betaine levels during treatment of a patient
with oral betaine in doses of between 20 and 120 mg/kg.204 They
found that serum levels of total homocysteine decreased
proportionally until betaine levels reached 400 mM, and suggested
that this is the therapeutic threshold for serum betaine.
Many authors5,12,196,202 have stressed the importance of early
diagnosis and therapy because of the poor prognosis in this
disorder once there is evidence of neurologic involvement. Even
with early diagnosis, it is not clear that any of the therapeutic
regimens are universally successful, and it is possible that genetic
heterogeneity in the disease itself is responsible for some of the
variability in clinical response to therapy.
Genetics. Autosomal recessive inheritance of methylene-H4Folate
reductase de®ciency was supported clinically by the occurrence of
more than one case in several families, by the presence of both
males and females with the disease within the same family, and by
the decreased activity of the enzyme in the ®broblasts165 and
lymphocytes167 of obligate heterozygotes. Consanguinity has been
reported.5,169
The gene is on chromosome 1p36.3 and has 11 exons.74
Although nonsense and splice-site mutations have been reported in
patients with methylene-H4Folate reductase de®ciency, most
mutations have been missense, and each has been reported in
only one or two families with severe de®ciency.203,210,220,247 Over
20 different mutations causing severe disease are known, in
addition to the polymorphisms described above, which may
contribute to disease in the general population; they are shown in
Fig. 155-5 in a representation of the methylene-H4Folate reductase
protein. The crystal structure of the E. coli enzyme, which is
Fig. 155-5 The structures, domains, and mutations of the MTHFR
polypeptide. Amino acid changes are indicated above the protein;
base pair position is indicated below. An area of the polypeptide is
considerably smaller than the mammalian one because it lacks the
C-terminal adenosylmethionine-binding regulatory domain, has
been reported.75 Based on this, the possible effect of the common
A222V polymorphism on the quaternary structural stability of the
enzyme and the role of folates in stabilizing this structure were
rationalized.75 The effects of severe, disease-causing mutations
have not yet been accounted for in this model system.
Methylene-H4Folate reductase de®ciency has been diagnosed
or excluded prenatally by enzyme assay or by measurement of the
incorporation of labeled formate into methionine by cultured
amniotic ¯uid cells.182,188,190,205,248,249 Enzyme activity is detectable in normal chorionic villi.182,188 If both mutations segregating
in a family are known, molecular analysis allows for early prenatal
diagnosis.
Functional Methionine Synthase De®ciency
Three metabolic pathways intersect at methionine synthase: those
of folate, cobalamin (Cbl), and sulfur-containing amino acids.
Because de®ciency of the activity of this enzyme results in
diminished or absent methylcobalamin (MeCbl) synthesis, disorders affecting it have been considered and designated genetically
as Cbl metabolism defects (symbolized as cbl ), along with others
affecting both MeCbl and adenosylcobalamin (AdoCbl) synthesis
or AdoCbl synthesis alone (see cobalamin section below). These
designations have been reinforced by the use, in differential
diagnosis, of genetic complementation analysis between cell lines
from patients with defects in all aspects of Cbl metabolism.
Nevertheless, because methyl-H4Folate participates in the methionine synthase reaction and because methionine synthase de®ciency
signi®cantly affects folate metabolism, these defects can be
considered inborn errors of folate metabolism as well. Here, we
discuss the two that directly involve methionine synthase (complementation groups cblE (MIM 236270) and cblG (MIM 250940). In
the subsequent sections on Cbl metabolism, the others indirectly
affecting methionine synthase activity (cblC, cblD, and cblF ) will
be considered in detail. For a full discussion of sulfur amino acid
metabolism and transsulfuration pathways, see Chap. 88.
Clinical and Laboratory Findings. Patients from the cblE and
cblG complementation groups are very similar, both clinically and
enlarged in order to show all the amino acid changes. (Courtesy of R.
Rozen, McGill University )
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3908
PART 17 / VITAMINS
Table 155-1 Clinical and Laboratory Features of Patient with
Homocystinemia Due to Defects in MeCbl Synthesis*
Mutant Class
Finding
cblE
cblG
Megaloblastic anemia
Developmental retardation
Cerebral atrophy
Hypotonia
Feeding dif®culty
Lethargy
Seizures
Vision abnormalities
Skeletal abnormalities
3/4
4/4
3/4
2/4
3/4
2/4
2/4
2/4
0/4
10/10
10/10
7/10
8/10
8/10
7/10
9/10
3/10
1/10
*Ratios denote the number of patients showing a particular finding/total
nrmber of patients in each mutant class
S O U RC E : Compiled from published summaries.243,259
biochemically. Most patients so far reported with these disorders
presented in the ®rst few months of life with vomiting, poor
feeding, and lethargy. Hypotonia, seizures, and developmental
delay characterize their severe neurologic dysfunction. There are
reports on at least 12 cblE and 20 cblG patients.83,243,250 ±261 Table
155-1 summarizes some of the clinical ®ndings available from the
literature. The prevalence of neurologic signs and symptoms is
striking. One patient in the cblG group presented as an adult with
progressively impaired sensory responses and gait disturbances
and was initially diagnosed as having multiple sclerosis.250
Megaloblastic anemia and homocystinuria or homocystinemia
are generally present, and hypomethioninemia is often found.
Serum Cbl and folate concentrations are normal or elevated, and
methylmalonic aciduria is absent, except in one patient, in whom it
was a transient ®nding.262
Localization of Defects. The constellation of homocystinuria and
hypomethioninemia without methylmalonic aciduria suggested
strongly that these patients had isolated de®ciencies in the activity
of methionine synthase, either primary or secondary to abnormal
synthesis or utilization of MeCbl, its cofactor (see Fig. 155-10).
Studies of ®broblasts derived from several of these patients have
con®rmed this hypothesis. Incorporation of [14C]propionate into
macromolecules was normal, while incorporation of [14C]methylH4Folate was reduced to 5 to 35 percent of control (average: 15
percent),243 a value similar to that reported for patients from the
cblC group (see below). Genetic complementation analysis based
on [14C]methyl-H4Folate incorporation distinguished two complementation groups:263 cblE (index patient reported in reference
264) and cblG (index patient reported in reference 252).
Accumulation of Cbl by ®broblasts was normal or increased in
these groups, as was the fraction recovered as AdoCbl.243 In
contrast, the fraction identi®ed as MeCbl was much reduced.243
cblE. When methionine synthase activities were determined
under standard conditions in extracts of ®broblasts from cblE
patients, both holoenzyme and total enzyme were normal or
slightly reduced.243 Under suboptimal assay conditions, namely in
the presence of lower concentrations of reducing agents,
methionine synthase activities were less than those in controls.265
These ®ndings have led to the hypothesis that the cblE group has
defects in an enzyme required either to reduce Cbl so that it can
participate in the methionine synthase reaction or to maintain it in
its active reduced form [cob(I)alamin] on the methionine
synthase.265 For example, bacterial methionine synthase has
accessory reductase proteins266 that perform these functions.
Based on the sequences of these bacterial proteins, Gravel and
colleagues cloned a cDNA for a multifunctional human protein
called methionine synthase reductase. Sequence analysis of
cDNAs for this enzyme from patients in the cblE group has
revealed a number of likely deleterious mutations in this
gene.83,257 Figure 155-6 is a linear representation of the protein,
its cofactor-binding regions, and the localization of the known
mutations.
cblG. In the cblG complementation group, methionine synthase
activities are reduced even under optimal assay conditions,243
although some heterogeneity has been noted.255 This ®nding
suggests that patients in this group have primary defects in the
catalytic subunit of methionine synthase itself.263 Moreover, the
cblG group shows biochemical heterogeneity with respect to
binding cellular Cbl to methionine synthase. In extracts of cell
lines from most patients, about 75 percent of cellular Cbl migrated
at the position of methionine synthase during gel electrophoresis,
even though little of it was MeCbl. In a few cell lines (referred to
as cblG variants), however, no Cbl of any form migrated at this
position.256 This was highly suggestive of mutations in the Cblbinding domain of methionine synthase or absent methionine
synthase protein in these patients, strengthening the possibility that
the cblG group re¯ected primary de®ciencies in the methionine
synthase apoenzyme. Human methionine synthase has now been
cloned, and mutations likely to be deleterious have been found by
sequence analysis of cDNAs from patients in the cblG
group.85,86,267 Based on the crystal structures of the Cbl-binding
and adenosylmethionine-binding domains of E. coli methionine
synthase,89,90 one of the missense changes uncovered (P1173L)
appears to disrupt the adenosylmethionine binding site,267 while
two others, I881D267 and H920D,85 may affect the pocket in the
Cbl-binding domain that accommodates the dimethylbenzimidazole moiety of the cofactor (see cobalamin section). Interestingly,
Fig. 155-6 Mutations in the MSR polypeptide.
Their relation to the FMN, FAD and NADPH
binding sites are shown. (Modi®ed from Wilson
et al.257 Used with permission.)
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
the cblG variants so far examined all appear to be effectively null
for the methionine synthase protein, rather than Cbl-binding
mutants.260
Pathophysiology and Genetics. The association of isolated
functional methionine synthase de®ciency with megaloblastic
anemia and neurologic defects in these patients provides further
strong evidence for the hypothesis outlined in Chap. 94, that these
clinical signs are sequelae of defects in the MeCbl-methionine
synthase branch of Cbl metabolism, rather than the AdoCblmethylmalonyl CoA mutase one. Hall268 and Shevell and
Rosenblatt269 have discussed in detail the relationships between
the biochemical defects and their pathophysiologic consequences.
As Hall has pointed out, there are three levels at which the impact
of these disorders is felt: hematologic, short-term neurologic, and
long-term neurodevelopmental. Effects at each of these may result
from a different aspect of functional methionine synthase
de®ciency, and the response of each to treatment may likewise
be distinctive. The hematologic problems may re¯ect disturbed
DNA synthesis, while the short-term neurologic symptoms are
likely due to either acute toxic effects or aberrant neurotransmitter
metabolism.268 The long-term developmental effects of these
disorders appear to be related to defects in myelination in the
central nervous system. Abnormal CT scans have been reported
for most of the patients on whom the test was performed, with
apparent atrophy or hypoplasia of the brain. MRI has been done
for only a few patients; in two, myelination was delayed, even after
a year of steady clinical improvement with Cbl therapy.268 Hall
has suggested a wide range of possibilities for the disruption of
function in the nervous system. They include toxicity of methylH4Folate or homocysteine, the classic folate trap hypothesis or
variants of it (discussed above), and reduced methylation of
proteins and neurotransmitters due to de®ciency in S-adenosylmethionine synthesis. DNA methylation, either from S-adenosylmethionine or directly from MeCbl,270 may also play a role. A
more complete understanding of the impact of de®ciencies in this
complex system of interrelated pathways on hematologic and
neurologic development requires further study, both of the
enzymes involved and of the patients with these and related
disorders.
Each of these diseases is inherited as an autosomal recessive
trait, with about equal numbers of male and female patients
reported.243,257,268 Both defects act as recessives in complementation analysis in culture.263 So far, heterozygote detection is
possible only by DNA-based techniques.
Diagnosis, Treatment, and Prognosis. The clinical hallmarks of
these disorders appear to be developmental delay and megaloblastic anemia, with homocystinuria and without methylmalonic
aciduria. Although most patients were diagnosed early in life, one
(a cblG) did not come to medical attention until age 21.250
Differentiation from other possible diagnoses such as TC II
de®ciency (see below), other folate transport or metabolism
defects, or cystathionine b-synthase de®ciency (Chap. 88) can be
accomplished by studies of cultured cells, particularly by
incorporation of 14C from [14C]methyl-H4Folate and complementation analysis. These two assays are especially important because
de®ciency of methylene-H4Folate reductase has a similar clinical
presentation and may result in decreased cellular MeCbl
accumulation and even decreased methionine synthase activity.208
Prenatal diagnosis using amniotic ¯uid cells is possible and has
been performed for both the cblE and cblG disorders. With the
cloning of both methionine synthase reductase and methionine
synthase, molecular diagnosis may be possible in families where
the mutations are known.
Because the patients reported to date have responded to
hydroxocobalamin (OH-Cbl) therapy with normalization of their
biochemical parameters and at least partial resolution of their
clinical symptoms236,243,255,268 and because it seems likely, as in
cblC patients (below), that delays in treatment may result in
incompletely reversible developmental delays or neurologic
de®cits,243,250,268 institution of OH-Cbl administration should
occur as soon as the diagnosis is made. Dosages of 1 mg OH-Cbl
per day (intramuscular injection) have been used initially, then
tapered to 1 mg, one to three times a week. Biochemical
improvement has been rapid on this regimen, and most clinical
symptoms have resolved in a few weeks. In some patients,
macrocytic anemia has responded to folinic acid therapy.259 In
general, neurologic symptoms and developmental problems have
been slower to improve, sometimes requiring 3 or 4 months of
therapy before consistent gains are apparent.243,268 On the other
hand, the cblE patient diagnosed prenatally and treated with OHCbl both in utero and postnatally has developed normally with
only minor clinical symptoms,269,271 suggesting that prenatal
therapy may be warranted in these disorders.
As in the case of some cblC patients (below), a variety of
adjuncts to Cbl therapy have been tried, including supplementation
with betaine, methionine, carnitine, and pyridoxine,269 with
variable and poorly documented results. Of these, betaine
supplementation to normalize the serum methionine:homocysteine
ratio further, beyond what is achieved with OH-Cbl alone, may be
justi®ed to avoid the vascular injury and thromboembolism
associated with homocystinemia (see Chap. 88). One patient in
the cblE group (diagnosed postmortem) died at 5 years of age
from bilateral renal artery thrombosis and had arteriosclerotic
changes elsewhere at autopsy,243 emphasizing the potentially
serious consequences of untreated or poorly controlled homocystinemia.
Because patients with these disorders have been described
relatively recently, the long-term prognosis in these conditions
remains unknown. The index cblE patient is 18 years old and
thriving, although he is mildly developmentally delayed,236 while
his prenatally diagnosed and treated brother (14 years old) appears
normal, except for a slight speech impediment.269 In contrast, the
index cblG patient, although clinically well, remains signi®cantly
retarded with major visual defects.252 It seems likely that patients
in the cblE and cblG groups will show a range of clinical
outcomes,243,268 similar to those of patients in the cblC group
(below), because the majority of the symptoms of all of these
patients arises from the same cause, that is, functional methionine
synthase de®ciency. Likewise, early diagnosis and treatment may
be the only way to avoid permanent neurologic damage and its
consequences.243,268
Differential Diagnosis of Folate Disorders
A guide to the differential diagnosis of the well-characterized
disorders of folate metabolism is shown in Table 155-2. Many of
these disorders are associated with normal serum and red blood
cell folate levels. Hereditary folate malabsorption is always, and
methylene-H4Folate reductase de®ciency is usually, associated
with low serum folate levels. Serum folate levels were reported as
elevated in most of the original Japanese patients (but none of the
subsequent ones) with glutamate formiminotransferase de®ciency.
Homocystinuria has been described in methylene-H4Folate
reductase de®ciency and in the cblE and cblG disorders.
Megaloblastic anemia is seen in hereditary folate malabsorption
and in cblE and cblG patients, but not in glutamate formiminotransferase de®ciency, except in the original Japanese patients, and
only rarely in methylene-H4Folate reductase de®ciency.
Defects detectable in cultured cells include a decreased
incorporation of label from methyl-H4Folate into protein or from
formate into methionine in cblE and cblG disease, and a decreased
content of methyl-H4Folate in ®broblasts from patients with
methylene-H4Folate reductase de®ciency. Cells from patients with
cblE and cblG show decreased levels of MeCbl, as may cells from
some patients with methylene-H4Folate reductase de®ciency.208 In
cell extracts from cultured ®broblasts, activity of methyleneH4Folate reductase is decreased in methylene-H4Folate reductase
de®ciency. In extracts of cblE and cblG cell lines, abnormalities in
methionine synthase activity can be detected. Abnormalities of
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3910
PART 17 / VITAMINS
Table 155-2 Inherited Defects of Folate Metabolism
Clinical ®ndings
Prevalence
Megaloblastic anemia
Developmental delay
Seizures
Speech abnormalities
Gait abnormalities
Peripheral neuropathy
Apnea
Biochemical ®ndings
Homocystinuria/hyperhomocysteinemia
Hypomethioninemia
Formiminoglutamic aciduria
Folate absorption
Serum Cbl
Serum folate
Red blood cell folate
Defects detectable in cultured
whole cells ®broblasts
Methyl-H4Folate ®xation
Methyl-H4Folate content
MeCbl content
Extracts-speci®c activity
Methionine synthase
holoenzyme
Glutamate
formiminotransferase
Methylene-H4Folate
reductase
Treatment
Hereditary
Folate
Malabsorption
MethyleneH4Floate
Reductase
De®ciency
Glutamate
Formiminotransferase
De®ciency
Methionine
Synthase
Reductase
De®ciency
(cblE)
Methionine
Synthase
De®ciency
(cblG)
< 20 cases
A
A
A
N
N
N*
N
> 40 cases
N
A
A
N
A
A
A
< 20 cases
N*
N*
N*
A*
N*
N*
N*
12 cases
A
A
A
N
N
N
N*
20 cases
A
A
A
N
A*
A*
N
N
N
A*
A
N
A
A
A
A
N
N
N
A
A*
N
N
A
N
N*
N*
N*
A
A
N
N
N
N
N
A
A
N*
N
N
N
N
N
N
N
N
A
N*
N
N
N
A
N
A
A
N
A
N
N*
N
N**
A
Activity undetectable in cultured cells
? Abnormal in liver and erythrocytes
N
Folic acid,
reduced folate
A
Betaine,
folate, methionine
N
?Folate
N
N
OH-Cbl, betaine,
reduced folate
*ˆ exceptions described in some cases.
**ˆ abnormal activity with low concentrations of reducing agent in assay.
N ˆ normal; A ˆ abnormal (i.e., clinical findings or laboratory findings present).
glutamate formiminotransferase have not been detected in any
cultured cell system.
COBALAMIN (VITAMIN B12)
The structure and function of cobalamins have intrigued students
of human biology since Minot and Murphy demonstrated that oral
administration of crude liver extract was effective in the treatment
of pernicious anemia in 1926.272 In 1948, this ``antipernicious
anemia factor'' was isolated from liver and kidney273,274 and was
named ``vitamin B12.'' De®ciency of the vitamin leads to an
alteration of function or morphology of several organ systems:
megaloblastic anemia and defective granulocyte and immune
system function; abnormal intestinal function; and neurologic
disease, including neurologic degeneration and dementia. Administration of as little as 1 mg of the vitamin daily was shown to
prevent relapse of pernicious anemia. Although the vitamin is
widely distributed in animal tissues, there is strong evidence that it
is synthesized only by microorganisms found in soil and water or
in the rumen and intestine of animals. See Dolphin275 and
Banerjee276 for comprehensive reviews of cobalamin structure,
biosynthesis, and chemistry.
Structural Features
The isolation of vitamin B12 culminated in the elucidation of its
three-dimensional structure by Hodgkin and coworkers using
x-ray crystallographic techniques.277 Cobalamin (Cbl), as it is now
of®cially designated, is composed of a central cobalt atom
surrounded by a planar corrin ring, which has a complex side
chain extending down from the corrin plane consisting of a
phosphoribo-5,6-dimethylbenzimidazolyl group (Fig. 155-7). One
of the nitrogens of the benzimidazole group is linked to the cobalt
atom by coordination in the ``bottom''(a) axial position. The
molecule is completed by coordination in the ``upper''(b) axial
position of several different radicals. Thus, cyanocobalamin (CNCbl) (more strictly, a-(5,6-dimethylbenzimidazolyl)-cobamide
cyanide) is formed by the complexing of a cyanide ion to the
cobalt atom. Although this compound is the most common
commercial form of the vitamin, it is an artifact of isolation and
does not occur naturally in microorganisms, plants, or animal
tissues. Many other Cbls have been formed with other ligands, but
only four have been routinely isolated from mammalian tissue:
hydroxocobalamin (OH-Cbl), the ``natural'' form of the vitamin,
glutathionylcobalamin (GSCbl), methylcobalamin (MeCbl), and
adenosylcobalamin (AdoCbl). Complexes of Cbl with other
sulfhydryl compounds have also been reported. MeCbl and
AdoCbl are unique for two reasons. They are the only two
compounds in nature known to have a direct covalent carboncobalt bond, and they are the only two forms of Cbl known to act
as speci®c coenzymes in mammalian systems.
Oxidation and reduction of the cobalt atom further complicate
the structure and nomenclature of the Cbls. In OH-Cbl, the cobalt
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
methyltetrahydrofolate (methyl-H4Folate), as well as methionine
synthase and MeCbl.286,287 It is relevant to the manifestations of
Cbl de®ciency and to the interrelationships between folate and
Cbl, and is discussed in detail in the folate section above.
The conversion of methylmalonyl CoA to succinyl CoA and
the methylation of homocysteine to methionine are the only Cbldependent reactions that have been demonstrated conclusively in
mammalian systems. Poston reported that AdoCbl acts as a cofactor in the enzymatic reaction by which a-leucine is isomerized
to b-leucine,288 but this has not been con®rmed in other
laboratories. In microorganisms, several other enzymes require
AdoCbl289,290 such as: glutamate mutase, glycerol dehydratase,
ethanolamine ammonia-lyase, and ribonucleotide reductase. In
addition, MeCbl participates in the formation of methane and
acetic acid and in the fermentation of lysine in bacteria.
Cobalamin Absorption and Distribution
Fig. 155-7 The structure of cobalamin. R 5 2CH2CONH2;
R0 5 2CH2CH2CONH2; X 5 2OH (hydroxocobalamin), 2CN (cyanocobalamin), CH3 (methylcobalamin), or 50 -deoxy-50 -adenosyl (adenosylcobalamin). (Reproduced from Fenton and Rosenberg.343 Used
with permission of the publisher.)
atom is trivalent (cob(III)alamin), and this compound has been
called ``vitamin B12a.'' When the cobalt is reduced to a divalent
state (cob(II)alamin), it is called ``vitamin B12r,'' and in the
monovalent state (cob(I)alamin), it is called ``vitamin B12s.'' These
oxidation-reduction states are important because the cobalt atom
must be reduced to its monovalent state prior to formation of
MeCbl or AdoCbl, apparently by speci®c reductase enzymes that
sequentially convert cob(III)alamin to cob(I)alamin, with cob(II)
alamin as an intermediate.278
Cobalamin Coenzymes
In 1958, Barker and his colleagues demonstrated that the
glutamate mutase reaction in Clostridium tetanomorphum required
vitamin B12,279 and, more speci®cally, that the active coenzyme
form of the vitamin was AdoCbl.280,281 One year later, Smith and
Monty reported that the analogous isomerization of methylmalonyl CoA to succinyl CoA was defective in the liver of Cbl-de®cient
rats.282 They suggested that Cbl was a cofactor for the latter
isomerization system, a thesis borne out by Gurnani et al.283 and
Stern and Friedmann,284 who showed in vitro that the activity of
methylmalonyl CoA mutase in liver from Cbl-de®cient animals
could be restored to normal by addition of AdoCbl, but not by CNCbl or other vitamin B12 analogues. For several years, because
AdoCbl was the only known coenzyme form of vitamin B12, it was
designated ``coenzyme B12.'' In 1966, Weissbach and colleagues285 demonstrated that MeCbl is a cofactor in the complex
reaction by which homocysteine is methylated to form methionine
(Fig. 155-8). This reaction requires S-adenosylmethionine and N 5-
Cbls have a unique and highly specialized mechanism of intestinal
absorption that has been reviewed in detail.236,291,292 The ability to
transport physiological quantities of vitamin depends on the
combined action of gastric, ileal, and pancreatic components (Fig.
155-9). The gastric substance, called ``intrinsic factor''(IF) by
Castle,293 who ®rst demonstrated its existence, is a glycoprotein
that binds Cbl in the intestinal lumen. IF, which has been isolated,
characterized extensively,291 and cloned,294 is synthesized by
gastric parietal cells. Evidence obtained in vitro295,296 and in
vivo297 suggests that three events precede the formation of IF-Cbl
in the gut lumen. First, Cbls are released from dietary protein in
the acid environment of the stomach. Second, Cbls bind to ``Rbinders,'' or haptocorrins, which are proteins of salivary and
gastric origin; these R-binders are members of a family of
glycoproteins with a high af®nity for Cbl. Third, pancreatic
proteases digest the R-binders, thereby liberating Cbls in the upper
small intestine, where they form complexes with IF. Subsequently,
the IF-Cbl complex interacts through its protein moiety with a
speci®c ileal receptor protein, called cubilin, in the presence of
calcium ions. The IF-Cbl-cubilin complex is recognized by
megalin, a general transport receptor, and transported into the
enterocyte by an endocytic mechanism; the complex is dissociated; and the vitamin is transported across the basal membrane
into the portal blood, bound to transcobalamin II (TC II), the
transport protein for newly absorbed vitamin.292 Evidence from
cultured adenocarcinoma cells, which behave like polarized
intestinal epithelial cells,298,299 suggests that these latter steps
re¯ect true apical-to-basal transcytosis in which the Cbl is bound
to newly synthesized TC II when it is released from the basolateral
membrane.299
When labeled Cbl is administered intravenously, most of the
labeled vitamin is immediately bound to TC II and disappears
from the plasma in a few hours.300,301 Only a small fraction binds
to transcobalamin I (TC I) or transcobalamin III (TC III) serum
glycoproteins of the R-binder family, even though they carry the
majority of the steady state serum Cbl.302 The Cbl bound to the Rbinders turns over very slowly, and its physiological role is still
unclear. MeCbl is the major circulating Cbl species, accounting for
60 to 80 percent of total plasma Cbl; OH-Cbl and AdoCbl make up
the remainder.303 Because > 90 percent of total plasma Cbl is
bound to TC I, it is clear that most of the circulating MeCbl travels
with this R-binder. This Cbl distribution pattern is puzzling,
particularly in the face of evidence indicating that AdoCbl
accounts for 70 percent of total hepatic Cbl, whereas MeCbl
constitutes a mere 1 to 3 percent.303 This preponderance of
AdoCbl is also present in other tissues, such as erythrocytes,
kidney, and brain. The physiological signi®cance of these widely
different fractional Cbl distributions in extracellular and intracellular compartments remains obscure.
TC II facilitates Cbl uptake by mammalian tissues. Finkler and
Hall304 showed that CN-Cbl bound to TC II was accumulated by
HeLa cells much more rapidly than free CN-Cbl or CN-Cbl bound
to TC I, IF, or other binding proteins. Such TC II-mediated uptake
3911
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3912
PART 17 / VITAMINS
Fig. 155-8 Reactions catalyzed by cobalamin
coenzymes in mammalian tissues. Note the
speci®city of adenosylcobalamin for the isomerization of methylmalonyl CoA and of
methylcobalamin for the methylation of homocysteine. Me-H4Folate 5 N 5-methyltetrahydrofolate; H4Folate 5 tetrahydrofolate.
was subsequently con®rmed in a variety of cell types, both in vivo
and in culture (liver; kidney; heart; spleen; lung; small intestine;
cultured ®broblasts; Chinese hamster ovary cells; mouse L cells;
lymphoma cells; and phytohemagglutinin-stimulated lympho-
Fig. 155-9 Pathway for the gastrointestinal absorption of dietary
cobalamin. Cbl 5 cobalamin; HC 5 haptocorrin (transcobalamin I/
III); HC-Cbl 5 haptocorrin-bound cobalamin; degHC 5 degraded
haptocorrin; IF 5 intrinsic factor; IF-Cbl 5 intrinsic factor-bound
cobalamin; -IF-Cbl 5 intrinsic factor-bound cobalamin attached to
cubilin, the ileal receptor; TCII-Cbl 5 cobalamin bound to transcobalamin II. (Reproduced from Rosenblatt and Fenton.477 Used with
permission.)
cytes) (see reference 292 for review). These ®ndings, coupled
with the observations in vivo that TC II disappeared from plasma
as TC II-Cbl was absorbed305 and appeared in lysosomal fractions
of hepatic306 and kidney cells,307 led to the proposal that the
circulating TC II-Cbl complex is recognized by a speci®c, widely
distributed plasma membrane receptor. This hypothesis has been
supported by considerable experimental evidence. Using 125Ilabeled TC II-Cbl complexes, Youngdahl-Turner and associates308
showed that the complex binds to a speci®c, high-af®nity (Ka
1010 M 1) cell-surface receptor on cultured skin ®broblasts
through a membrane site that recognizes TC II and by a
mechanism dependent on Ca2‡. They showed further that the
TC II-Cbl complex is then internalized intact via adsorptive
endocytosis309 and that the degradation of TC II and release of Cbl
from the complex occur as a result of lysosomal protease
activity.308,309 Cbl then exits from the lysosome by a mediated
process,310 and is either converted to MeCbl bound to the
methionine synthase in the cytosol or enters the mitochondrion,
where, after reduction and adenosylation to AdoCbl, it is bound to
methylmalonyl CoA mutase.311,312
The intricate process just described is the most widely
distributed physiological means by which mammalian cells obtain
Cbl, but it is not the only one. Hepatocytes, for instance, contain a
surface receptor for asialoglycoproteins, and this receptor interacts
with TCI-Cbl (and perhaps TC III-Cbl) complexes, thereby
providing a second potential means by which this particular tissue
obtains Cbl.313 There is also evidence that at least some tissues are
capable of taking up free (unbound) Cbl, if the unbound vitamin is
raised to suf®ciently high concentrations. In cultured ®broblasts,
this uptake process for free Cbl is saturable, Ca2‡-independent,
and sensitive to inhibitors of protein synthesis and sulfhydryl
reagents.314 Its functional role, under most circumstances, is
probably negligible.
Coenzyme Biosynthesis and Compartmentation
Because methylmalonyl CoA mutase, the mammalian enzyme
dependent on AdoCbl, is a mitochondrial protein,315 whereas the
MeCbl-dependent methionine synthase is cytoplasmic,316 it is
important to relate the cellular biology of the vitamin to its cellular
and molecular chemistry. The chemical pathway of AdoCbl
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
Fig. 155-10 General pathway of the cellular uptake and subcellular compartmentation of cobalamins, and of the intracellular
distribution and enzymatic synthesis of cobalamin coenzymes. TC II 5 transcobalamin II; OH-Cbl 5 hydroxocobalamin;
MeCbl 5 methylcobalamin; AdoCbl 5 adenosylcobalamin; CblIII,
CblII, CblI, 5 cobalamins with cobalt valences of 13, 12, and 11,
respectively.
synthesis was de®ned initially in bacteria.278,317 Three enzymes
are required for coenzyme synthesis, two reductases and an
adenosyltransferase. The reductases are ¯avoproteins that require
NAD as a cofactor. The ®rst (EC 1.6.99.8) is responsible for
converting cob(III)alamin, for example, OH-Cbl, to cob(II)alamin,
and the second (EC 1.6.99.9) is responsible for catalyzing the
further reduction to cob(I)alamin. The latter compound and ATP
are substrates for an adenosyltransferase (EC 2.5.1.17) that
completes the synthesis of AdoCbl. Neither of the reductases
has been puri®ed extensively, but the adenosyltransferase has. It
has a pH optimum of 8, requires Mn2‡, and has a Km of
110 5 M for cob(I)alamin and 1.610 5 M for ATP.317 The
biosynthetic steps leading to de novo MeCbl formation are not as
clear. Because maintenance of MeCbl on methionine synthase
requires a reductase system to generate cob(I)alamin on
methionine synthase itself, this reduction-methylation sequence
seems likely for de novo MeCbl synthesis as well.318,319
Accumulated evidence indicates that mammalian cell metabolism of Cbl proceeds by a very similar set of reactions (Fig.
155-10). In 1964, Pawalkiewicz et al.320 showed that human liver
and kidney homogenates could convert CN-Cbl to AdoCbl.
Several years later, AdoCbl synthesis from OH-Cbl was observed
in HeLa cell extracts incubated with ATP and a reducing system
that presumably bypassed the enzymatic reduction of OH-Cbl
(cob(III)alamin) to cob(I)alamin.321 Subsequently, Mahoney and
Rosenberg322 demonstrated the synthesis of both AdoCbl and
MeCbl by intact human ®broblasts growing in a tissue culture
medium containing OH-[57Co]Cbl. This system was subsequently
characterized in cell extracts.323,324 As with the HeLa cell system,
chemical reductants were employed to bypass both cobalamin
reductases.324 Such extracts synthesized AdoCbl, thereby demon-
strating that a homologue of the adenosyltransferase found in
bacteria also exists in normal human cells. These experiments also
revealed that the adenosyltransferase was mitochondrial in
location, implying that both the synthesis and cofactor activity
of AdoCbl take place in this organelle. The reductive steps in
mammalian systems are still poorly understood. Pezacka and
colleagues have suggested that GSCbl may be an intermediate in
these reactions.325 Watanabe et al. demonstrated both microsomal
and mitochondrial reductase activities,326 but suggested that one or
more of these may be nonspeci®c activities of other enzymes, such
as the cytochrome b5-cytochrome b5 reductase complex.327 It
seems certain, as shown in Fig. 155-10, that MeCbl synthesis takes
place in the cytosol in conjunction with the methionine synthase
and methionine synthase reductase (see ``Folate'' section above).
Metabolic Abnormalities in Cobalamin De®ciency
The biochemical abnormalities in plasma and urine of patients
with Cbl de®ciency re¯ect the dysfunction of the enzymes
dependent on Cbl coenzymes. The ®rst relevant observation in
this context was Cox and White328 and Barness and his
colleagues,329 demonstration that methylmalonic acid excretion
in the urine was distinctly increased in Cbl-de®cient patients with
classic pernicious anemia. The methylmalonic aciduria in these
patients was reversed rapidly by administration of physiologic
doses of Cbl, indicating that repletion of Cbl restored the
methylmalonyl CoA mutase reaction to normal. Later, Cox et al.
reported that patients with Cbl de®ciency also had distinctly
increased amounts of propionic acid in the urine, this abnormality
again being reversed by treatment.330 Interestingly, they also found
excessive amounts of acetic acid in the urine of Cbl-de®cient
subjects. The mechanism leading to this abnormality is not clear
3913
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3914
PART 17 / VITAMINS
because acetate does not participate in the major pathway of
propionate catabolism. The ®nding could, of course, re¯ect
increased utilization of the alternative pathways of propionate
metabolism in the face of a block in the major pathway, because
each alternative route leads eventually to the formation of acetyl
CoA (see Chap. 94). Excessive excretion of homocystine has also
been documented in Cbl-de®cient patients,331,332 as has combined
methylmalonic aciduria and homocystinuria. Allen and his
colleagues examined a large series of patients with suspected or
proven Cbl de®ciency333,334 and showed that 95 percent of them
have methylmalonic acidemia, homocystinemia, or both, often
without hematologic signs. A number of reports now document the
occurrence of Cbl de®ciency in vegetarian and macrobiotic
communities, with accompanying metabolic derangements.335,336
In some cases, clinical symptoms have been observed as well,
particularly in breast-fed offspring of strict vegetarian mothers,
who are themselves de®cient in the vitamin.337,338
Cobalamins and Folic Acid
An interesting, important, and still puzzling aspect of Cbl function
concerns its relationship to folic acid.339 Several lines of evidence
bear out this relationship: the appearance of megaloblastic anemia
in either Cbl or folate de®ciency; the reversal of megaloblastic
anemia in Cbl de®ciency by large doses of folate; the amelioration
of megaloblastic changes in folic acid de®ciency by pharmacologic doses of CN-Cbl; the increased plasma concentrations of
methyl-H4Folate in patients with cobalamin de®ciency; the
excretion of excessive amounts of FIGLU after histidine loading
in patients with either Cbl or folate de®ciency; and the reduced
amounts of total Cbl in the liver of patients with folate de®ciency.
A plausible explanation for most of these effects was proposed
independently by Herbert,340 Noronha,341 and Larrabee342 and
their colleagues, and has been referred to as the folate trap
hypothesis. This thesis rests on the evidence that the conversion of
methyl-H4Folate to H4Folate depends on the MeCbl-dependent
reaction in which homocysteine is methylated to methionine. If
methionine biosynthesis is the only quantitatively signi®cant
reaction using methyl-H4Folate, Cbl de®ciency will interfere with
the folate cycle and, barring other control mechanisms, will lead to
the accumulation of methyl-H4Folate and the depletion of other
folate derivatives. This depletion could become severe enough to
interfere with other reactions requiring H4Folate, such as the
synthesis of purines or pyrimidines and the conversion of FIGLU
to glutamate. Under these circumstances, H4Folate de®ciency
could be relieved by administration of either folates or Cbl, but
only the latter would complete the folate cycle. This scheme, if
totally correct, would obviate the need for additional Cbldependent mechanisms to explain the megaloblastic changes
observed in Cbl de®ciency and would account for the speci®c
disorders of folate metabolism observed in Cbl-de®cient
human beings. It does not explain the low Cbl content of
livers from folate-de®cient subjects or the hematologic response
of folate-de®cient patients to Cbl (also see ``Folate'' section
above).
INBORN ERRORS OF COBALAMIN
TRANSPORT AND METABOLISM
Inherited disorders in the transport and metabolism of Cbl
manifest themselves clinically in ways that re¯ect the underlying
defect and, in particular, that depend on which coenzyme is
de®cient and, hence, which of the two Cbl-dependent enzymes is
reduced in activity (see Fig. 155-8). Defects that affect only
AdoCbl biosynthesis generally lead to metabolic ketoacidosis in
the newborn or infant period, and regularly result in methylmalonic acidemia and methylmalonic aciduria. MeCbl de®ciencies
present as failure to thrive, megaloblastic changes, and neurologic
signs, usually with homocystinuria and hypomethioninemia.
De®ciencies of both coenzymes produce a variable combination
of these signs and symptoms.
Combined AdoCbl and MeCbl De®ciency
Cbl was ®rst described as ``extrinsic factor,'' an antipernicious
anemia factor found in aqueous extracts of raw liver, which
combined with ``intrinsic factor'' (IF), a component of normal
gastric secretions, to cure pernicious anemia, an acquired disease
resulting from gastric insuf®ciency. It is recognized, however, that
there are several inborn errors of metabolism with presentations
similar to pernicious anemia that result from abnormal Cbl
transport or from altered cellular Cbl metabolism. Although these
diseases share the general clinical phenotype of failure to thrive,
developmental delay, neurologic dysfunction, and megaloblastic
anemia, the details of their presentations allow them to be
differentiated. (For reviews, see references 236, 269, 292, 302,
and 343).
Transport Defects: Clinical and Laboratory Findings
Food Cobalamin Malabsorption. Carmel and colleagues
described a number of patients, mostly adults, with a condition
that includes low serum Cbl concentrations in the face of a normal
Schilling test.344 Neurologic manifestations, with or without
megaloblastic changes, appear to be common.344 These individuals suffer from an inability to release Cbl from the proteinbound state in which it is normally encountered in foodstuffs,
usually measured by the absorption of Cbl bound to egg yolk.345
Because this process requires both an acid gastric pH and peptic
activity,346 any underlying factors that compromise gastric
function, including atrophic gastritis or partial gastrectomy, can
result in this disorder.347 However, a signi®cant fraction of patients
have shown no evidence of impaired gastric function,344 suggesting that a more subtle mechanism, whose nature is currently
unknown, may be responsible.348
Intrinsic Factor (IF) De®ciency. A number of children have been
described with a juvenile form of pernicious anemia (see reference
349 for references to case reports). The clinical symptoms, which
usually appear after the ®rst year and before the ®fth year of age,
include developmental delay and the megaloblastic anemia
characteristic of pernicious anemia.236,349 Serum levels of Cbl
are markedly de®cient, but, in contrast to the adult disease, gastric
function and morphology are normal, and serum autoantibodies to
IF are absent. Cbl absorption is abnormal in these children, but is
restored when the vitamin is mixed with normal human gastric
juice as a source of IF. Further investigations of the gastric
secretions of these patients have shown that, as expected, they
suffer from one of several different classes of functional IF
de®ciency. One results in failure to produce or secrete any
immunologically recognizable IF,350,351 while another causes
production of immunologically reactive protein that is inactive
physiologically.349,352±354 The latter group includes patients
whose IF has reduced af®nity for the ileal IF receptor,353 reduced
af®nity for Cbl,355 or increased susceptibility to proteolysis.349 In a
few cases with partial de®ciency, presentation was delayed into the
second decade or later.352,356 Although a cDNA for human
intrinsic factor has been characterized ([GIF], NM_005142, MIM
261000), no mutations have yet been described.357
Enterocyte Cbl Malabsorption (Selective Vitamin B12 Malabsorption, MGA1, Imerslund-GraÈsbeck Syndrome) (MIM
261100). More than 250 cases of a related disorder have been
described with the clinical signs of juvenile pernicious anemia, but
with normal IF and normal gastrointestinal function, except for
speci®c intestinal Cbl malabsorption.358± 361 In addition to
megaloblastic anemia and serum Cbl de®ciency, many of these
patients have proteinuria. Similarly to IF de®ciency, patients
usually present between 1 and 5 years of age, although some have
been diagnosed much later.362 In contrast to IF de®ciency patients,
however, these children's Cbl absorption defect is not corrected by
providing normal human IF with the vitamin.236 There has been a
decrease in the number of new cases in recent years, and it has
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
been suggested that there may be dietary or other factors
modifying expression.363
Most patients are found in Norway, Finland, Saudi Arabia, and
among Sephardic Jews. Using microsatellite markers in Finnish
and Norwegian families, the disease gene was mapped to a 6-cM
region on chromosome 10p12.1.363 An IF-Cbl-binding protein
called cubilin ([CUBN], NM_001081, MIM 602997) was puri®ed
from renal proximal tubule, cloned, and localized to the same
region.364,365 This large (400 kDa) protein comprises a short
N-terminal domain, eight EGF repeats, and 27 contiguous, 110amino acid CUB domains, ®rst identi®ed in certain developmental
control proteins. Two mutations in the cubilin gene (CUBN) have
been found segregating in Finnish families with this disorder.366
One, P1297L, is found in homozygous form in most Finnish
patients (31 of 34 alleles). It changes a highly conserved proline
residue, which is predicted by x-ray structural analysis of other
CUB domains367 to be part of a ligand-binding site. Interestingly,
this change is in CUB domain 8, suggested by deletion and
expression studies to be part of the IF-Cbl binding site (domains 5
to 8).368 The other change, homozygous in one patient, is a point
mutation in an intron in CUB domain 6, resulting in missplicing,
insertions containing stop codons in mRNA, and truncated
predicted gene products. No cubilin was detected on western
blots of proteins from this patient's urine, establishing clearly that
CUBN is the gene for the IF-Cbl receptor protein.368 Neither of
these mutations has been found in non-Finnish families, and the
mutations causing this disorder in other populations are being
sought.
Cubilin is a peripheral membrane protein with no clearly
de®ned transmembrane domain. It copuri®es with megalin, an
even larger receptor protein of the LDL receptor gene family, and
colocalizes with it in both intestinal and renal epithelium.364,369
Megalin appears to be a transporter for many proteins370 and may
be responsible for the endocytosis of the cubilin-IF-Cbl complex.
Cubilin itself may have other functions, as suggested by its speci®c
binding of receptor-associated protein368 and its function in
apolipoprotein A-I endocytosis.371
Earlier experiments suggested that, in at least some patients,
the ileal receptor for IF-Cbl was normal as measured by IF-Cbl
binding to homogenates of ileal biopsy specimens.360 In others, a
functional receptor appeared to be absent.372 Because the
mechanism for Cbl transport across the enterocyte is complex, it
seems possible that this syndrome encompasses defects at several
points in this overall pathway, including cubilin itself (as shown in
the Finnish pedigrees), cubilin internalization via megalin, or Cbl
transfer to TC II within the enterocyte (also see discussion of cblF
below). It remains to be seen how many patients with this
phenotype will be found to have mutations in the cubilin gene
itself.
Transcobalamin II (TC II) De®ciency. At least 36 cases have
been described of de®ciency of TC II, including both twins and
sibs.236,373 In contrast to the previous two disorders, TC II
de®ciency has generally presented within the ®rst or second month
of life as failure to thrive, with such nonspeci®c symptoms as
vomiting and weakness, accompanied by megaloblastic anemia
and, eventually, immunologic de®ciency and neurologic disease.236,373 Because the patients may have immature white blood
cell precursors in a marrow that is otherwise hypocellular, they
have been misdiagnosed with leukemia. Neurologic disease is not
present at the time of diagnosis, but may develop with an extended
duration of illness, inadequate cobalamin treatment, or treatment of
the anemia with folates and not cobalamin.373 Interestingly, serum
Cbl levels are normal or nearly so in these patients, re¯ecting the
fact that most serum Cbl is carried by TC I and other R-binders.302
It is essential to measure blood levels of TC II before the patient
has been started on cobalamin therapy. Intestinal Cbl absorption
has been abnormal in some patients, but not in others.236
Most patients have had no immunologically detectable TC II in
plasma,236 although a few had detectable protein,374,375 and at
least one produced a TC II that was able to bind Cbl, although
apparently without function.374 In those patients who do not
synthesize TC II, both the diagnosis and prenatal diagnosis can be
performed by studying the ability of cultured ®broblasts or
amniocytes to synthesize TC II.376
Treatment of TC II de®ciency requires that serum cobalamin
levels be kept very high. It is important to monitor levels carefully
and to ensure that the patient is compliant, particularly if oral
treatment is used. Serum levels ranging from 1000 to 10,000 pg/
ml have been required; these levels have been achieved with oral
OH-Cbl or CN-Cbl in doses of 0.5 to 1.0 mg twice weekly or by
weekly doses of 1 mg OH-Cbl. Although folic acid or folinic acid
can reverse the megaloblastic anemia, folate should never be given
as the only therapy because of the danger of hematologic relapse
and neurologic deterioration.
The human TC II gene is on chromosome 22, a cDNA has been
cloned, and the molecular basis of some variants determined
([TCN2], NM_000355, MIM 275350).377± 380 In a number of
patients who have no TC II synthesis, deletions and nonsense
mutations have been found.381,382
R-Binder De®ciency ([TC I], NM_001062, MIM 193090). Several individuals are known who have de®cient or absent R-binder
(TC I) in plasma, saliva, and blood cells.236,383 Although these
patients have serum Cbl values in the de®cient range, they show no
signs of Cbl de®ciency, probably because their TC II-Cbl levels
are normal. Although several of these patients have had a
myelopathy not attributable to other causes,383,384 the etiology of
these symptoms remains unclear, emphasizing our lack of
understanding of the role of R-binders in normal Cbl metabolism
and homeostasis. It should be noted that R-binders carry Cbl in
mother's milk, so the potential exists for Cbl de®ciency in breastfed infants of mothers with this de®ciency.
Chemical Abnormalities and Pathophysiology. While megaloblastic anemia is the hallmark of the Cbl transport disorders, the
chemical abnormalities expected to accompany functional Cbl
de®ciency have also been found in many cases. In theory, Cbl
de®ciency should lead to de®cient synthesis of both AdoCbl and
MeCbl and, thus, to decreased activities of their respective
enzymes, resulting in methylmalonic acidemia(-uria) and homocystinemia(-uria). When examined carefully, most patients with
each of the transport de®ciencies have these chemical symptoms,
although the quantities of both methylmalonate and homocystine
excreted have generally been much lower than in patients with
abnormalities in cellular Cbl metabolism (see below). On the other
hand, some patients do not have one or either of these chemical
abnormalities.236 To a certain extent, these variable ®ndings,
which do not appear to correlate well with the nature of the defect
or the severity of the general symptoms and hematologic
aberrations, may result from the fact that alternative pathways of
Cbl transport exist that, although minor in normal individuals, may
contribute signi®cantly in patients with these transport defects. For
example, receptors for free Cbl have been found on HeLa cells,385
human ®broblasts,314 and adenocarcinoma cells,299 and may
permit some Cbl transport even in the absence of one of the
transport proteins. In addition, hepatocytes may be able to recover
some Cbl from asialo-TC I-Cbl by means of the asialoglycoprotein
receptor system.386 This could be particularly important in TC II
de®ciency.
One major clinical difference between the two intestinal
transport defects and TC II de®ciency lies in the different age of
onset of these conditions. While neither intestinal Cbl transport
de®ciency manifests itself before 1 year of age, many TC IIde®cient patients are symptomatic within 1 or 2 months of birth,
with some exceptions (see above). This appears to be due to two
factors. First, the IF-dependent pathway for intestinal Cbl
absorption may not become important until later in infancy,
when the gastrointestinal tract switches from pinocytotic mechanisms of transport to receptor-mediated ones. IF-Cbl transport falls
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3916
PART 17 / VITAMINS
into the latter category. Interestingly, one report387 demonstrates
that, in rats, expression of IF by the gastric mucosa increases
abruptly from the low levels found in the newborn animal to adult
levels at about the time of weaning (13 to 20 days), consistent with
this hypothesis. Second, the body stores considerable amounts of
Cbl beyond daily requirements in blood, liver, and other tissues.
Thus, IF-de®cient patients and those with Imerslund-GraÈsbeck
syndrome likely show no signs of de®ciency early in infancy both
because the IF-dependent mechanism for Cbl transport is not yet
operating and because they have acquired suf®cient Cbl through
other mechanisms or prenatally to sustain themselves for a time
after the developmental switch in intestinal absorption has
occurred. Conversely, because TC II is presumably necessary for
ef®cient Cbl transport into cells regardless of the mechanism by
which it is acquired, TC II-de®cient patients have no symptomfree period and become ill as soon as their maternally derived
stores of Cbl are exhausted. The observation that most TC IIde®cient patients have been normal at birth likely re¯ects the fact
that fetal tissues concentrate Cbl relative to the maternal serum.388
It is not certain why the neurologic manifestations of TC II
de®ciency are less severe than those found in the inborn errors of
cobalamin metabolism that affect cofactor synthesis.373
The megaloblastic anemia characteristic of these disorders
likely re¯ects a de®ciency in the activity of methionine synthase
brought about by the absence of its cofactor, MeCbl. Because
patients with isolated de®ciency of AdoCbl (see below) or its
partner enzyme, methylmalonyl CoA mutase (see Chap. 94), are
usually hematologically normal, this conclusion seems to be solid.
Likewise, the severe neurologic manifestations,373 particularly in
patients who are diagnosed long after the onset of their disease,
appear more likely to be due to de®cient methionine synthase
activity. Although isolated mutase de®ciency can produce central
nervous system dysfunction (see Chap. 94), this is believed to be at
least partially a consequence of the severe metabolic ketoacidosis
experienced by these patients, a condition not generally present in
patients with Cbl transport defects.
The speci®c etiology of the hematologic and neurologic
disturbances in these individuals is not completely understood,
but clearly it must derive from the central role of methionine
synthase in cellular 1-carbon metabolic pathways, both in terms of
folate metabolism (see sections above) and homocysteinemethionine balance (see Chap. 88). Because the folate cycle in
mammalian cells requires that methyl-H4Folate transfer its methyl
group to homocysteine, via MeCbl, in order to regenerate
tetrahydrofolate, it has been suggested that the accumulation of
methyl folate in the absence of the Cbl coenzyme serves as a folate
trap, which produces functional folate de®ciency intracellularly.340,342 The effects of this de®ciency on the important roles
that folate metabolism plays in the synthesis of nucleotides and,
hence, of RNA and DNA could easily account for the general
disruptions in cellular homeostasis in rapidly dividing tissues, such
as the hematopoietic system. Whether this folate trap hypothesis is
equally applicable to explaining the neurologic dysfunction in
these patients is not clear. An alternative explanation might
involve disruption of the interconversion of homocysteine and
methionine and of S-adenosylhomocysteine and S-adenosylmethionine and interference with the role these compounds play
in methylation and enzyme regulation in the central nervous
system.270,389 Until more is known about these pathways, however,
either hypothesis will be dif®cult to establish.
Genetics and Molecular Biology. Each of the genetic lesions in
Cbl transport appears to be inherited as an autosomal recessive
trait on the basis of classic genetic criteria.236 Because the
Imerslund-GraÈsbeck syndrome may actually encompass de®ciencies in more than one protein (receptor) (see above), it remains
possible that other modes of inheritance exist for a subset of
families with this disorder. Rat and human IF cDNAs have been
cloned,294,390 and the IF gene has been localized to human
chromosome 11.294 There is suggestive evidence from biochem-
ical analysis of some presumptive heterozygotes for IF de®ciency
that they express both a normal and an abnormal allele for IF and
for the idea that some patients with IF de®ciency express two
different mutant alleles.349 The rat IF cDNA has been used to
establish that only one cell type Ð the chief cell of the rat gastric
mucosa Ð expresses IF in the adult animal,387 in keeping with
immunochemical studies that indicate that the parietal cell is the
only source of IF in human beings and other mammals.291 As
mentioned above, both cubilin and megalin have been cloned, and
deleterious mutations in cubilin have been described in Finnish
Imerslund-GraÈsbeck patients. TC II has electrophoretic isoforms
in normal individuals,391 and it has been suggested that some cases
of TC II de®ciency manifest themselves as abnormal isoforms.392
The structural locus for TC II is on the long arm of chromosome
22,294,393 linked to the P blood group system locus. Although TC I
has been cloned,394 mutations in it leading to R-binder de®ciency
have not been described.
Diagnosis and Treatment. The Cbl transport de®ciencies, except
for food Cbl malabsorption, are usually diagnosed initially by the
observation of the combination of macrocytic anemia with
developmental delay or failure to thrive.236 Neurologic symptoms
may be present at later times.373 Serum Cbl levels are low in food
Cbl malabsorption, IF de®ciency, and Imerslund-GraÈsbeck syndrome, but usually normal in TC II de®ciency. Schilling tests are
normal in the ®rst disorder, abnormal in the second two, and may
also be abnormal in some cases of TC II de®ciency.236 The second
two disorders can be differentiated by determining whether the
Schilling test becomes normal when the test Cbl is incubated with
normal human IF before it is administered. Only IF de®ciency
patients show correction. Con®rmation of either of the intestinal
malabsorption defects entails demonstration of normal gastric and
ileal function other than the speci®c Cbl absorption de®ciency, the
absence of antibodies to IF, and, in some cases of IF de®ciency, the
absence of functional (i.e., Cbl-binding) or immunologically
cross-reacting IF in the patient's gastric secretions.236,349 TC II
de®ciency can sometimes be differentiated from the other two by
an age of onset within the ®rst months (as opposed to years) of life.
The diagnosis can often be established by measuring the
unsaturated Cbl-binding capacity of the patient's serum; in normal
individuals, this largely re¯ects the amount of TC II present. Gel®ltration chromatography can be used to separate TC II from
serum R-binders and thus provides a more accurate assessment of
Cbl-binding capacity. Unfortunately, both these tests can be
compromised by previous Cbl therapy, possibly even by previous
Schilling tests.395 Because TC II is synthesized by many cell types,
including ®broblasts, and because ®broblasts from TC II-de®cient
patients synthesize a defective protein or none at all,314 a more
satisfactory approach may be to grow patient ®broblasts in
medium without TC II and to then determine whether any
functional TC II has been synthesized by incubating the cells with
radiolabeled Cbl and measuring the extent to which TC II-Cbl
accumulates in the medium or in the cells.314,376
In the case of sibs or other relatives in families in which one of
these defects has been diagnosed, hematologic changes can
provide an early sign of the presence of the disease, as can
methylmalonic acidemia and homocystinemia. In the two
intestinal transport disorders, Schilling tests may prove abnormal
before the onset of clinical symptoms. In TC II de®ciency, because
cord blood contains fetal, not maternal, TC II,396 it is possible to
test immediately for the presence of functional TC II. Biochemical
prenatal diagnosis is possible only for TC II de®ciency, based on
the ability of normal amniocytes to synthesize functional TC II.376
Because no fetus at risk for TC II de®ciency has yet been tested by
this method and predicted to be affected, its applicability remains
hypothetical. The two intestinal malabsorption syndromes are not
expressed in accessible fetal tissues (if, in fact, those proteins are
expressed at all during fetal life) and, thus, cannot be diagnosed
prenatally by a biochemical test. The cDNA cloning of IF294 and
cubilin364,365 may make a DNA-based diagnostic procedure, such
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
Table 155-3 Clinical and Laboratory Features of Patients with Methylmalonic
Acidemia and Homocystinuria*
Mutant Class
Finding
Clinical
Sex (male/female)
Failure to thrive
Developmental retardation
Seizures
Feeding dif®culties
Hypotonia
Microcephaly
Nystagmus
Hydrocephalus
Dementia
Myelopathy
Laboratory
Normal serum cobalamin
Hematologic abnormalities
Acidosis
Microthrombi
Pigmentary retinopathy
Decreased visual acuity
cblC
Early Onset
cblC
Late Onset
21/44
26/44
21/44
32/44
27/44
19/44
10/44
8/44
1/44
1/44
1/6
2/6
2/6
3/6
3/6
0/6
0/6
0/6
3/6
3/6
44/44
31/44
9/44
3/44
19/44
15/44
6/6
3/6
0/6
0/6
1/6
0/6
cblD
cblF
2/0
0/2
1/2
0/2
0/2
2/4
5/6
5/6
1/6
3/6
3/6
2/2
0/2
0/6
1/2
3/6
3/6
*Ratios (except for sex) denote the number of patients showing a particular finding/total number of
patients in each mutant class; empty cells indicate data were not available.
S O U RC E : Information obtained from published surveys,420 case reports,413,417±419,425 and personal
communications.
as RFLP analysis, possible in at least some cases involving
de®ciency of these proteins.
The major treatment regimen for each of these disorders has
been pharmacologic doses of Cbl, either CN-Cbl or OH-Cbl,
usually administered by injection to avoid dependence on gastric
factors and ileal uptake of free Cbl.236,395 Titration of the dosage
used and frequency of therapy should be carried out to ensure
resolution of all clinical abnormalities, particularly in TC IIde®cient patients with defective immune system function397,398 or
neurologic disorders.373 The serum Cbl concentration at which
patients become asymptomatic has varied widely, especially in TC
II de®ciency, and should be used as a guide only after the patient
has been stabilized. Folate has been administered to some TC IIde®cient patients with effective correction of the hematologic
signs of the disease.236 At least one of these patients suffered a
relapse, however, and the ability of folate to resolve other
symptoms, particularly long-term neurologic dysfunction, has not
been determined. Consequently, folate therapy should only
accompany effective doses of Cbl.373
The prognosis in these diseases appears to be generally very
good as long as serum Cbl levels are maintained appropriately.373,399 Interestingly, while Cbl therapy has been effective in
normalizing the hematologic signs in Imerslund-GraÈsbeck
patients, the proteinuria often observed in this disorder has been
unchanged even by many years of therapy.399 Although one
woman with TC II de®ciency has borne two normal children,375 it
remains unclear whether Cbl therapy can achieve complete
reversal of the neurologic damage and developmental delay that
occur if patients remain undiagnosed for an extended period (or if
adequate Cbl levels are not maintained) or whether some residual
de®cit may persist in these cases.373
Defects in Cellular AdoCbl and MeCbl Synthesis
Clinical and Laboratory Findings. In comparison to the Cbl
transport defects described above, defects in the cellular
metabolism of Cbl generally result in clinically more severe
metabolic disease. As a consequence, patients with these disorders
regularly show the metabolic disturbances that result from
de®cient synthesis of both AdoCbl and MeCbl, namely methylmalonic acidemia and homocystinuria (Fig. 155-8). Because the
amounts of these metabolites detected in these patients generally
greatly exceed those found in patients with Cbl transport defects or
Cbl de®ciency, their measurement has served to distinguish these
groups of individuals clinically. We are aware of over 100 patients
with inherited combined methylmalonic acidemia and homocystinuria. Many of the early patients were the subjects of individual
case reports.400± 413 Cells from these children comprise three
biochemically and genetically distinct complementation groups,
designated cblC (MIM 277400), cblD (MIM 277410), and cblF
(MIM 277380).323,414± 416 The cblC group is by far the largest
(more than 100 patients), with the cblD represented by 2 sibs400
and the cblF group by 6 unrelated individuals.413,417±419
cblC. Clinical ®ndings have varied widely among patients in the
cblC group; Table 155-3 presents a summary of the clinical and
laboratory data from a survey of 50 such patients.420 Most of the
early described patients presented in the ®rst few months of life
because of failure to thrive, poor feeding, or lethargy. Subsequent
reports have emphasized that some patients have a much delayed
onset of symptoms: for example, a 4-year-old with fatigue,
delirium, and spasticity,406 and a 14-year-old with the rather
sudden onset of dementia and myelopathy.405 Thus, regardless of
age, neurologic manifestations have been prominent. Most, but not
all, of these patients have had hematologic abnormalities
characterized by megaloblastoid and macrocytic anemia; hypersegmented polymorphonuclear leukocytes and thrombocytopenia
have been observed less often. Several patients have had features
of hemolytic-uremic syndrome.421 A few patients have had a
characteristic pigmentary retinopathy with perimacular degeneration, as well as other ophthalmologic changes.404,406,409,412
Hydrocephalus, cor pulmonale, and other congenital malformations also have been seen.422± 424 Moderate to severe developmental delay has been common in the early onset patients, and
about a third of early onset patients have died despite treatment.420
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3918
PART 17 / VITAMINS
In addition to the methylmalonic aciduria and homocystinuria that
characterize this group of patients, they have hypomethioninemia
and cystathioninuria. The methylmalonic aciduria in these children
is distinctly less severe than that encountered in children with
isolated mutase de®ciency (see Chap. 94), although much more
severe than that reported for patients with Cbl transport defects.
Moreover, neither hyperglycinemia nor hyperammonemia has
been reported in any of the cblC (or cblD or cblF ) patients. Serum
cobalamin and folate concentrations have been normal.
cblD. In sharp contrast with this description, neither of the
brothers in the cblD group400 had any clinically signi®cant
problems until much later in life. The older brother came to
medical attention because of severe behavioral pathology and
moderate mental retardation at 14 years of age. He had, as well, a
poorly de®ned neuromuscular problem involving his lower
extremities. His then 2-year-old brother was asymptomatic,
although biochemically affected. No hematologic abnormalities
have been noted in either sib.
cblF. There have been six unrelated patients reported in the cblF
group.413,417± 419,425 The ®rst two girls presented during the ®rst
weeks of life with stomatitis and hypotonia, together with minor
facial anomalies. The ®rst patient, who was not diagnosed until 8
months, developed poorly and was clearly delayed.413 No
hematologic abnormalities were found in the ®rst patient, while
the second showed macrocytosis and hypersegmented polymorphonuclear neutrophils.425 The second patient died suddenly at
home. Other cblF patients have had pancytopenia, neutropenia, or
thrombocytopenia.417,418 Although the ®rst patient had no
detectable homocystinuria despite cellular de®cits in methionine
synthase activity and in MeCbl synthesis, all the others have
shown both methylmalonic aciduria and homocystinuria. A male,
diagnosed at 11 years of age, had a grossly abnormal Schilling test
and low serum Cbl.417 He had recurrent stomatitis in infancy,
arthritis at age 4 years, and confusion and disorientation at age 10
years. He also had a pigmentary skin abnormality.
Localization of Cellular Metabolism Defects
cblC and cblD. It is clear that patients in the cblC and cblD
groups have a defect in cellular metabolism of Cbl based on
several criteria: total Cbl content of liver, kidney, and cultured
®broblasts is markedly reduced;401,426±428 the ability of cultured
cells to retain [57Co]-labeled CN-Cbl429 or to convert [57Co]labeled CN-Cbl or OH-Cbl to AdoCbl and MeCbl is markedly
impaired;430 activities of methylmalonyl CoA mutase and
methionine synthase in cultured cells are de®cient, such de®ciency
being partially reversed by supplementation of the growth medium
with OH-Cbl;415,431,432 and the mutase and the methionine
synthase apoenzymes in cells from affected patients appear to be
normal.84,400,415,431
Because these mutant cells demonstrate normal receptormediated adsorptive endocytosis of the TC II-Cbl complex and
normal intralysosomal hydrolysis of TC II,274,309,415,433 perusal of
Fig. 155-10 makes it clear that the defects in the cblC and cblD
cells must affect some step or steps subsequent to cellular uptake,
common to the synthesis of both coenzymes, and prior to the
binding of the Cbl coenzymes to their respective apoproteins.
Signi®cantly, cblC (and, to a lesser extent, cblD) cells use CN-Cbl
less well than OH-Cbl432,434 and are unable to convert CN-Cbl to
OH-Cbl, a step shown in normal cells to be a metabolic
prerequisite for the synthesis of both AdoCbl and MeCbl.434 The
latter results have been interpreted as evidence for a defect in a
cytosolic cob(III)alamin reductase, which is required for reducing
the trivalent cobalt prior to alkylation.434 Partial de®ciencies of
CN-Cbl b-ligand transferase and of microsomal cob(III)alamin
reductase have been described in cblC and cblD ®broblasts.435,436
A partial de®ciency of mitochondrial NADH-linked aquacobalamin reductase was described by Watanabe and his coworkers in
two cblC ®broblast extracts.437 The suggestion by Pezacka and her
colleagues that GSCbl may be the product of an intermediate step
in this process325 provides another potential site for the mutation
in one of these groups. Finally, it should be mentioned that the
distinction between the cblC and cblD classes is based ®rst and
foremost on complementation studies that de®ne the two classes as
unique.415 Their biochemical differences appear to be quantitative
rather than qualitative, with the cblC group having more severe
metabolic derangements than the siblings designated cblD.
Therefore, it remains possible that the cblD mutation is allelic to
cblC and shows interallelic complementation.
cblF. Studies using cultured ®broblasts from two patients in the
cblF group413,416,425 are of particular interest. As with cells from
cblC and cblD patients, both mutase and methionine synthase
activities were impaired, and AdoCbl and MeCbl contents were
reduced. In contrast to the cblC and cblD mutants, however, the
cblF cells accumulated unmetabolized, nonprotein-bound CN-Cbl
in lysosomes.438,439 These ®ndings indicate that cblF cells are
de®cient in the mediated process by which Cbl exits from
lysosomes after being taken up by receptor-mediated endocytosis.310 Two brief reports further indicate that two cblF patients had
abnormal Schilling tests with both free and IF-bound Cbl,417,440
suggesting that the putative lysosomal defect affects ileal Cbl
transcytosis as well (see Cbl transport section above).
Pathophysiology. The megaloblastic anemia so commonly
observed in the cblC patients almost surely re¯ects the disturbance
of methionine synthase activity. This can be stated with some
assurance because patients with isolated methylmalonyl CoA
mutase de®ciency (see Chap. 94) more severe than that
encountered in the cblC patients exhibit no megaloblastic anemia.
The early and severe central nervous system abnormalities
encountered in the cblC group probably re¯ect the methionine
synthase abnormality as well, in that such patients generally do not
experience the severe metabolic ketoacidosis that probably
accounts for the central nervous system problems in patients
with mutase de®ciency only. Thus, patients with severe, inherited
dysfunction in the synthesis of both Cbl coenzymes resemble
closely patients with exogenous Cbl de®ciency Ð both groups
having prominent hematologic and neurologic manifestations
resulting from the blocked methionine synthase system.
Genetic Considerations. Because equal numbers of affected
males and affected females exist in the cblC group, because
females have been as seriously affected as males, and because cells
from affected patients behave as recessives in complementation
studies,414 it seems safe to predict that this disorder is inherited as
an autosomal recessive trait. The mode of inheritance of the cblD
and the cblF mutations cannot yet be de®ned, because of the
paucity of known patients (both affected cblD patients in the only
family yet described are male); both males and females have been
identi®ed in the cblF group. Identi®cation of heterozygotes for the
cblC, cblD, or cblF group has not yet been accomplished.
Diagnosis, Treatment, and Prognosis. The combination of
methylmalonic aciduria and homocystinuria with normal serum
Cbl concentrations and normal TC II is the set of biochemical
parameters needed to distinguish patients in the cblC, cblD, and,
probably, cblF groups from those with methylmalonic acidemia
caused by isolated methylmalonyl CoA mutase de®ciency (see
Chap. 94); from those with homocystinuria due to cystathionine
synthase de®ciency (see Chap. 88), or methylene-H4Folate
reductase de®ciency, or isolated methionine synthase de®ciency
(see ``Folate'' section above); and from those with Cbl transport
defects or exogenous Cbl de®ciency (see above). It should be
noted that one cblF patient had low serum Cbl when diagnosed.417
Because each of the cellular metabolic defects is expressed in
cultured cells from affected individuals, the diagnosis should be
con®rmed by genetic complementation analysis between patient
®broblasts and ®broblasts from patients whose complementation
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
Table 155-4 Salient Biochemical Features of Cultured Fibroblasts from Patients with
Various Defects in Cellular Cbl Metabolism*
Mutant Class
Biochemical Parameter
Studies with intact cells
[14C]propionate oxidation
[14C]Methyl-H4Folate ®xation
MeCbl synthesis
AdoCbl synthesis
Conversion of CN-Cbl to OH-Cbl
Lysosomal ef¯ux of free Cbl
Enzyme activities in cell extracts{
Mutase holoenzyme
Mutase total enzyme
Methionine synthase holoenzyme
Methionine synthase total enzyme
Methionine synthase reductase
Cob(I)alamin adenosyltransferase
cblA
cblB
cblC
cblD
cblE
cblF
cblG
±
‡
‡
±
‡
‡
±
‡
‡
±
‡
‡
±
±
±
±
±
‡
±
±
±
±
‡
‡
±
±
‡
‡
‡
±
±
±
±
±
±
‡
±
±
‡
‡
‡
±
‡
‡
‡
‡
‡
±
‡
‡
‡
‡
±
±
‡
±
‡
‡
±
‡
±
‡
‡
nt
nt
**
‡**
±
nt
nt
nt
±
‡
‡
nt
nt
±
±
‡
nt
*‡ ˆ normal; ± ˆ markedly deficient or undetectable; ˆ partially deficient; nt ˆ not tested.
{Holoenzyme is defined as that enzyme activity measured in the absence of added cofactor; total enzyme
is that activity measured in the presence of saturating concentrations of cofactor.
**Activity dependent on reducing conditions used (see folate section).
Abbreviations: Methyl-H4Folate ˆ N 5-methyltetrahydrofolate; MeCbl ˆ methylcobalamin; AdoCbl ˆ
adenosylcobalamin; CN-Cbl ˆ cyanocobalamin; OH-Cbl ˆ hydroxocobalamin.
groups have been determined previously. This technique also
allows the cblC, cblD, and cblF groups to be distinguished from
each other. Biochemical studies on cultured cells, such as Cbl
uptake, lysosomal Cbl ef¯ux, or AdoCbl and MeCbl synthesis, and
direct measurement of mutase and methionine synthase activities
in cell extracts can be performed to provide further con®rmation
(see Table 155-4).
Because normal amniotic ¯uid cells appear to carry out all the
steps of Cbl metabolism observed in cultured ®broblasts, it is be
possible to detect each of these defects prenatally by assaying any
of these parameters in cultured amniocytes. This has been carried
out successfully in both the cblC and cblF269 groups.
The distinctions between these cellular metabolic defects and
other related conditions are critically important, because appropriate therapy and prognosis depend on them. Whereas exogenous
Cbl de®ciency responds dramatically to physiologic amounts of
Cbl and transport defects to somewhat larger dosages, successful
management of cblC, cblD, and cblF demands the administration
of large amounts of OH-Cbl (up to 1 mg daily) by intramuscular
injection.195,400,402,404,406,417,440 Such treatment has resulted in
dramatic decreases in urinary methylmalonate and in less
dramatic, but signi®cant, decreases in urinary homocystine in
many patients who have received it. The form of Cbl administered
is important, at least in cblC patients, because studies on cultured
cells from this group have shown that supplementation in culture is
much less ef®cient with CN-Cbl than with OH-Cbl in eliciting an
increase in the activity of the affected enzymes.438 A recently
published study of the effects of the chemical form of Cbl
administered to two cblC patients supports the greater ef®cacy of
OH-Cbl, both biochemically and clinically.441 A number of
adjunctive therapies have been employed for cblC patients with
variable success, including moderate protein restriction to reduce
the load of metabolic end products and, hence, the amount of
methylmalonate produced; carnitine supplementation, to improve
organic acid excretion and relieve a postulated functional carnitine
de®ciency (see Chap. 94); folic and folinic acid administration, to
bypass the so-called methylfolate trap and restore hematologic
function; and betaine administration, to provide substrate for
betaine:homocysteine methyltransferase, which is not dependent
on a Cbl coenzyme, and thus to return the serum methionine:homocysteine ratio toward normal. Few investigators have evaluated
the ef®cacy of these treatments critically, however. Bartholomew
and his colleagues attempted to determine the effects of OH-Cbl
dosage schedule and of treatment with carnitine, folinic acid, and
betaine on the clinical and biochemical status of two patients with
the cblC defect.442 In each case, the OH-Cbl injection schedule
could be titrated to control the patient's methylmalonic acidemia
and homocystinuria. In addition, betaine administration (250 mg/
kg/day) appeared to act synergistically with the OH-Cbl to
produce a further reduction in plasma homocystine. No speci®c
clinical improvement accompanied the betaine therapy, however.
Neither patient responded clinically or biochemically to folinic
acid or carnitine treatment. The overall result in both patients was
good metabolic control, as measured by reduced methylmalonic
acidemia and normal serum homocysteine and methionine
concentrations, and resolution of most of their clinical symptoms,
such as lethargy, irritability, vomiting, and failure to thrive, with a
treatment regimen of daily betaine administration and biweekly
injections of OH-Cbl. Signi®cantly, both patients remained
somewhat delayed developmentally, even after a year or more of
therapy. In addition, the retinal degeneration present in these
patients was not reversed by the therapy, although some
improvement in cone response was noted in one of them.
This report serves also to emphasize that early diagnosis and
prompt institution of therapy with OH-Cbl (and possibly betaine)
may be the only way to change the outcome of these patients,
which, at least in the case of the cblC group, is dismal thus far
(Fig. 155-11). Many have died despite intensive therapy. Severe
hemolytic anemia is a major complication in the deceased cblC
patients, as has congestive heart failure. Thromboemboli, so often
encountered in patients with homocystinuria due to cystathionine
synthase de®ciency, have, thus far, been documented in only a few
cblC patients420 and in the older of the two cblD brothers, in whom
this complication was not noted until he reached 18 years of age.
Betaine treatment may reduce this risk by normalizing the serum
methionine/homocysteine ratio, even when Cbl-responsiveness is
incomplete.442 Surviving patients, even those under apparently
good metabolic control, continue to show signs of neurologic
dysfunction, including mild to moderate mental retardation and
delayed development of motor skills,236,420 and, in some cases, the
continued presence of abnormal ophthalmologic ®ndings.442
These problems could be the result of irreversible damage that
3919
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3920
PART 17 / VITAMINS
Fig. 155-11 Outcome in cblC patients based on age-of-onset less
than 1 year (early onset, shaded bars) or greater than 1 year (late
onset; solid bars). Late onset patients presented between ages 4 and
14 years. Impairment is classi®ed in functional terms as severe,
moderate, or mild according to the expectation for age. (Reproduced
from Rosenblatt et al.420 Used with permission.)
occurred prior to diagnosis and therapeutic intervention, or could
re¯ect the impossibility of completely correcting the cellular
lesion in Cbl metabolism in certain cells whose function is critical
in neurologic development. Signi®cantly, patients with apparently
later onset or milder disease have done considerably better (Fig.
155-11). Until a number of patients with these defects are
diagnosed before birth or soon thereafter, and treated immediately,
or even prenatally, with Cbl and betaine supplements, we will not
know whether the poor outcome in this group can be modi®ed
signi®cantly. Documentation of such experience is particularly
important in assessing the clinician's ability to modify the natural
history of these disorders.
Clinical and Laboratory Presentation. As mentioned above, the
clinical ®ndings in patients with methylmalonic acidemia due
either to defective mutase enzyme (mut) or defective AdoCbl
synthesis (cblA, cblB) are remarkable more for their similarities
than for their differences. A survey of the natural history in 45
such patients has been reported:451 20 were mut; 14 were cblA, and
11 were cblB (also see Chap. 94). There were approximately equal
numbers of males and females in each group. The most common
signs and symptoms at the onset of clinical dif®culty were lethargy,
failure to thrive, recurrent vomiting, dehydration, respiratory
distress, and muscular hypotonia. Little interclass difference was
observed for these major clinical manifestations or for such less
common ones as developmental retardation, hepatomegaly, or
coma. The only major clinical distinction between the mut group
and the groups with defective AdoCbl synthesis was that most of
the former group presented very early in life ( < 1 to 4 weeks),
while 60 percent of the cblA group and 45 percent of the cblB
group presented between 1 month and 1 year.451
The laboratory ®ndings in cblA and cblB patients at the time
that methylmalonic acidemia was ®rst documented are shown in
Table 155-5, with those in mut patients for comparison. As
expected, serum cobalamin concentrations were routinely normal.
Metabolic acidosis, with blood pH values as low as 6.9 and serum
bicarbonate concentrations as low as 5 mEq/liter, was observed in
the majority of patients. Ketonemia or ketonuria, hyperammonemia, and hyperglycinemia or hyperglycinuria were also observed
in many affected patients. Leukopenia, thrombocytopenia, and
anemia were the only other manifestations that were noted. Earlier
case reports (reviewed in reference 452) found that hypoglycemia
occurred in about 40 percent of affected patients. Signi®cantly, the
megaloblastic anemia characteristic of functional Cbl de®ciency or
the inherited disorders of MeCbl synthesis (cblC, cblD, cblE, cblF,
and cblG) was not present in these patients.
Defects in AdoCbl Synthesis
In 1968, Rosenberg443,444 and Lindblad445,446 and their colleagues
described infants with severe metabolic ketoacidosis and developmental delay who accumulated very large amounts of
methylmalonate in blood and urine, similar to patients reported
earlier by Oberholzer447 and Stokke448 and their coworkers. In
contrast to the earlier patients, however, these infants responded
dramatically to pharmacologic doses of CN-Cbl or AdoCbl with
resolution of their clinical symptoms and major reductions in their
excretion of methylmalonate. Further studies indicated that the
methylmalonyl CoA mutase enzyme was normal in these patients,
but that synthesis of AdoCbl was impaired.430,449 Somewhat later,
Kaye et al.450 reported two patients with methylmalonic acidemia
who were unresponsive in vivo to high doses of CN-Cbl but who
also had apparently normal mutase enzyme and defective AdoCbl
synthesis. Subsequent biochemical and genetic complementation
analysis established that lesions at two genetically distinct loci can
be responsible for defective AdoCbl synthesis; they are designated
cblA (MIM 251100) and cblB (MIM 251110)323,414 Because both
groups of patients with de®cient AdoCbl synthesis share many
clinical features with those with primary defects in the
methylmalonyl CoA mutase enzyme, the reader is also referred
to Chap. 94 for a discussion of the latter group.
Chemical Abnormalities In Vivo. Large amounts of methylmalonic acid have appeared in the urine or blood of all reported
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
Table 155-5 Laboratory Findings in 45 Patients with
Methylmalonic Acidemia*
Mutant Class
Finding at Clinical Onset
cblA
cblB
mut
Normal serum cobalamin
Metabolic acidosis
Ketonemia and/or ketonuria
Hyperammonemia
Hyperglycinemia and/or -glycinuria
Leukopenia
Anemia
Thrombocytopenia
100
100
78
50
70
70
10
75
100
88
67
83
83
45
45
45
100
89
88
76
60
69
44
40
*
Numerical values are the percentages of the patients in each group in whom
the particular finding was made.
S O U RC E : From Matsui et al.451
patients. Whereas normal children and adults excrete less than
0.04 mM (5 mg) methylmalonate daily, children with isolated
methylmalonic acidemia have excreted from 2.1 to 49 mM (240 to
5700 mg) in a 24-h period. Their plasma concentrations of
methylmalonate, almost undetectable in normal subjects, have
ranged from 0.22 to 2.9 mM (2.6 to 34 mg/dl). In the few patients
in whom it was measured, the CSF concentration of methylmalonate equaled that of plasma (see reference 392 for references to
early case reports). No relationship between the quantities of
methylmalonate accumulated in body ¯uids and the etiology of
mutase de®ciency (i.e., apoenzyme vs. coenzyme synthesis
de®ciency) has been reported. Methylmalonate is surely the major,
but not the only, abnormal metabolite found in body ¯uids of these
patients. Because propionyl CoA carboxylation is reversible,
propionate and some of its precursors (butanone) or metabolites
(b-hydroxypropionate and methylcitrate) also accumulate in blood
and urine,443,453,454 their amounts being small compared to that of
methylmalonate (see Chap. 94).
Several groups have studied the relationship between protein or
amino acid loading and methylmalonate accumulation in these
patients. Without exception, administration of protein or amino
acids known to be precursors of propionate and methylmalonate,
such as methionine, threonine, valine, or isoleucine, has resulted in
augmented methylmalonate accumulation and, in some instances,
ketosis or acidosis.443,445,447,448 When Cbl-responsive patients are
given supplements of this vitamin, such augmentation by
methylmalonate precursors is lessened considerably.455 All these
®ndings suggest that patients with discrete defects at the mutase
step have a major block in the utilization of methylmalonyl CoA,
which is expressed as methylmalonate accumulation.
Localization of Enzymatic Defects. Because the conversion of
propionate to succinate is blocked in each of the methylmalonic
acidemias, whether due to mutase defects or AdoCbl synthesis
de®ciencies, an early screening test for these disorders measured
the ability of intact peripheral blood leukocytes or cultured
®broblasts to oxidize [14C]propionate or [14C]methylmalonate to
14
CO2 and compared this with the oxidation of [14C]succinate to
14CO .444 More recently, incorporation of [14C]propionate into
2
trichloroacetic acid-precipitable material by intact cultured cells
has replaced the more cumbersome 14CO2 evolution technique.456,457 Further discrimination among the methylmalonic
acidemias has depended on studies of cobalamin uptake and
AdoCbl formation by intact cultured ®broblasts, on assays of
mutase activity in cell extracts, and on genetic complementation
studies with cultured cells.
cblA. A series of observations by Rosenberg449 and Mahoney430
and their colleagues on the ®broblasts of the index patient with
Cbl-responsive methylmalonic acidemia led to the demonstration
of a primary defect in AdoCbl synthesis. Such intact cells were
unable to convert OH-[57Co]Cbl to Ado[57Co]Cbl, although they
took up the labeled vitamin normally and had no abnormality in
synthesizing the other cobalamin coenzyme, MeCbl.430 On the
other hand, cell-free extracts from this line synthesized AdoCbl
normally when incubated with OH-[57Co]Cbl, ATP, and a reducing
system designed to bypass cob(III)alamin reductase and cob(II)alamin reductase and to measure only cob(I)alamin adenosyltransferase.323 Fibroblasts from other patients in this clinically de®ned
group had identical ®ndings in similar studies.323 Genetic
complementation analysis established unequivocally that all these
patients belonged to a single complementation group, designated
cblA, and thus presumably had defects in the same enzyme or
protein.414 Because it has been shown that intact mammalian
mitochondria can synthesize AdoCbl from OH-Cbl in vitro without
prior reduction458 and because Cbl adenosyltransferase activity is
normal in this group,323 it is presumed that the defect must lie in
one of the early steps of mitochondrial Cbl metabolism, possibly
in a mitochondrial Cbl reductase (see Fig. 155-10). So far, the lack
of a speci®c assay for the reductive step(s) in AdoCbl
synthesis326,327 has prevented a more precise localization of the
defect in the cblA group.
cblB. Another group of patients with defective AdoCbl synthesis
was uncovered when Cbl metabolism was examined in a number
of cell lines from patients with methylmalonic acidemia.323,459
Some of these ®broblasts showed a primary defect in AdoCbl
synthesis similar to that described for the cblA class (above),
except in one aspect. When cell-free extracts from these lines were
incubated with OH-[57Co]Cbl, a reducing system, and ATP, no
AdoCbl synthesis was detected,323 in contrast to the result in the
cblA cell lines. Because this assay is speci®c for ATP:cob(I)alamin
adenosyltransferase, the patients in this group must have defects in
this enzyme.324 Complementation analysis indicates that a single
locus, cblB, is involved in all these patients and that it is distinct
from the cblA locus.414
Pathophysiology. All studies in vivo and in vitro in patients with
methylmalonic acidemia due to methylmalonyl CoA mutase
de®ciency, either primary or secondary to AdoCbl synthesis
defects, indicate that the block in the conversion of methylmalonyl
CoA to succinyl CoA explains fully the accumulation of
methylmalonate in blood and urine; the augmentation of
methylmalonate excretion and the precipitation of ketosis by
protein, amino acids, or propionate; and the excretion of longchain ketones formed in the catabolism of branched chain amino
acids. See Chap. 94 for a complete discussion of methylmalonic
acidemia.
By comparing and contrasting the ®ndings in patients with
isolated mutase de®ciency, whether due to defects in mutase or in
AdoCbl synthesis, with those in patients with functional Cbl
de®ciency (as in classic pernicious anemia or the Cbl transport
defects discussed above), it is possible to shed some light on the
mechanism responsible for the hematologic and neurologic
abnormalities in the latter disorders. Thus, the absence of
megaloblastic anemia in any patient with isolated mutase
de®ciency militates against any involvement of this enzyme in
the typical megaloblastoid seen in Cbl de®ciency. Similarly, the
cerebellar and posterior column abnormalities so often encountered in Cbl-de®cient patients have never been observed in patients
with methylmalonic acidemia due to speci®c mutase dysfunction.
Therefore, the notion that neurologic dysfunction in pernicious
anemia re¯ects aberrant incorporation of odd-chain or branched
chain fatty acids into myelin because of a block in the propionate
pathway has little to recommend it. It appears likely, then, that
abnormalities in Cbl-dependent methionine synthase account for
the hematologic and neurologic abnormalities in Cbl-de®cient
patients.
Genetic Considerations. Both cblA and cblB are almost certainly
inherited as autosomal recessive traits. This conclusion is based on
3921
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3922
PART 17 / VITAMINS
these observations: (a) approximately equal numbers of affected
males and females have been reported in each group; (b) no
instance of vertical transmission from affected parent to affected
child has been reported; (c) each mutant class behaves as a
recessive in culture in complementation experiments;414,415,431
and (d) cell lines from heterozygotes for the cblB mutation show
partial adenosyltransferase de®ciency.324
Diagnosis, Treatment, and Prognosis. Because simple colorimetric assays for urinary methylmalonate and more complex gas
chromatography-mass spectrometry assays for serum and urinary
methylmalonate are available, it should not be dif®cult to make a
diagnosis of methylmalonic acidemia, once this condition is
considered. Other sources of neonatal or infantile ketoacidosis
must be ruled out. The quantity of methylmalonate excreted, the
absence of megaloblastic changes, and the normal amounts of
serum homocysteine, methionine, and Cbl all serve to differentiate
these diseases from others that may lead to methylmalonic
aciduria. Distinguishing between primary mutase de®ciency and
primary AdoCbl synthesis defects and between the two causes of
the latter ultimately depends on studies with cultured cells B
routinely, genetic complementation analysis.415 Prenatal detection
of methylmalonic acidemia has been accomplished in two
different ways: by measurement of methylmalonate in amniotic
¯uid and maternal urine at midtrimester460,461 and by studies of
mutase activity and Cbl metabolism in cultured amniotic ¯uid
cells.456,461,462 Assays of [14C]propionate utilization456 in uncultured chorionic villous biopsy specimens have proven unsatisfactory, however. AdoCbl synthesis defects of both complementation
groups461,462 have been identi®ed in these ways.
Two treatment regimens for children with methylmalonic
acidemia exist and should be employed in tandem for patients with
AdoCbl synthesis de®ciencies. A diet restricted in protein (or a
special formula restricted in amino acid precursors of methylmalonate) should be instituted as soon as such life-threatening
problems as ketoacidosis, hypoglycemia, or hyperammonemia
have been addressed; and supplementary Cbl (1 to 2 mg OH-Cbl
intramuscularly daily for several days) should be given as soon as
the diagnosis of methylmalonic acidemia is made (or even
seriously considered). Such measures should decrease the
circulating concentrations of methylmalonate and propionate.
Even Cbl-unresponsive children with delayed development
improve markedly when treated with careful dietary protein
restriction.463,464 In Cbl-responsive patients, titration of Cbl
dosage schedules against methylmalonate excretion and clinical
status is probably worthwhile. The methylmalonic aciduria is not
completely eliminated in even the most responsive patients, even
though clinical symptoms such as ketosis and acidosis are
completely resolved. As discussed in Chap. 94, Roe and
associates465±467 have pointed out that L-carnitine supplements
may be a useful therapeutic adjunct in patients with methylmalonic acidemia, presumably by repleting intracellular and extracellular stores of free carnitine that are depleted in affected
patients because of exchange with excess methylmalonyl CoA and
propionyl CoA. No trial of this compound has been reported in
cblA or cblB patients. As suggested in Chap. 94, oral antibiotic
therapy may prove useful as well. Thompson and his colleagues
reported that three Cbl-unresponsive patients showed subjective
improvement in alertness and appetite following brief metronidazole therapy;468 longer treatment periods have resulted in
signi®cant improvements in other patients, including decreased
number and severity of acidotic episodes, increased appetite,
decreased vomiting, growth acceleration, and improved behavior
in a cblB patient.469
The previously mentioned survey451 suggested that both the
response to Cbl supplements and the long-term outcome in
affected patients depends considerably on the nature of the
biochemical lesion. Whereas more than 90 percent of the cblA
patients responded to Cbl supplements with a distinct fall in blood
or urinary methylmalonate, only 40 percent of the cblB patients
showed such a response. Presumably, the 60 percent of cblB
patients unresponsive to Cbl supplements have such complete
adenosyltransferase de®ciency that AdoCbl synthesis cannot be
augmented by Cbl supplements, in distinction to the cblB patients
with apparently ``leaky'' mutations that permit responsiveness in
vivo. The uniform responsiveness of patients in the cblA group
suggests either that the responsible mutations are generally leaky,
thereby allowing mass action to result in more AdoCbl synthesis,
or that alternative pathways of Cbl reduction, which require high
substrate concentrations, exist in cells.326,327 As in the case of
primary mutase de®ciency, it should be emphasized that clinical
responsiveness in vivo does not require complete correction of the
functional mutase de®ciency or complete normalization of
biochemical parameters such as the methylmalonic acidemia
(see Chap. 94). Some patients in the cblB group, unresponsive to
CN-Cbl or OH-Cbl in vivo, might be expected to respond to
AdoCbl itself, but published reports on two patients suggest that
this logical alternative is ineffective.470,471 Unpublished experiments on cells in culture suggest that AdoCbl is largely converted
back to OH-Cbl during transport.
The long-term outlook for affected patients is revealing. The
cblA patients (i.e., the group biochemically most responsive to Cbl
supplements) had the best outcome according to the survey Ð 70
percent were alive and well at ages up to 14 years and presumably
continue to be so. The cblB group had about equal fractions in the
alive and well, the alive and impaired, and the deceased category.
It is interesting, albeit anecdotal, that the index patient in the cblA
group (now over 30 years old) discontinued Cbl supplements at
age 9 years despite advice to the contrary. In the ensuing years,
despite accumulation of very large amounts of methylmalonate in
the blood and urine, his development and general health have
remained excellent, with one exception (see below). Perhaps, as in
some other inherited metabolic disorders, treatment of methylmalonic acidemia is most critical early in life. If this experience is
borne out, it makes expert clinical management in the early weeks
or months of life most important. There have been several reports
of ``metabolic stroke'' in patients following episodes of metabolic
decompensation.472± 474 Three of the patients472,473 belonged to
the Cbl-responsive cblA group, but were not being treated at the
time. Extrapyramidal signs, particularly dystonia, were accompanied by bilateral lucencies of the globus pallidus and persisted
after the acute crisis had passed. In one case, the dystonia was
gradually progressive over a period of 7 years without visible
progression of the neurologic lesions.474 One complication of
long-term survival of some methylmalonic acidemia patients may
be chronic renal failure. One report has indicated that 8 of 12
nonresponsive patients (aged 1 to 9 years) had reduced GFR, with
5 severely affected.475 In one patient ``greatly improved metabolic
control'' over a period of 18 months led to increased, but still
impaired, renal function.475 Signi®cantly, the index cblA patient
referred to above recently returned to attention following treatment
for renal dysfunction due to biopsy-proven interstitial nephritis.
The impact of better metabolic control and Cbl supplementation
has not been explored in this case.
Finally, the feasibility of prenatal therapy with Cbl supplements has been demonstrated. Ampola et al.461 showed that
administration of Cbl supplements to a woman carrying an
affected fetus of the cblA group resulted in signi®cant reduction in
maternal excretion of methylmalonate and the presence of only
moderate methylmalonic acidemia(-uria) in the newborn child.
She was doing well at the time of the report (20 months) with
moderate protein restriction and occasional Cbl therapy, whereas
an undiagnosed affected sib had died at 3 months of age.461 A
second, similar case has been reported.476
SUMMARY
Tables 155-2 and 155-4 summarize the salient biochemical
features of patients with defects in various aspects of folate and
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CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM
Fig. 155-12 Summary scheme of inherited defects of cobalamin
metabolism. The circled numbers and their key signify the general
sites at which abnormalities have been identi®ed and the
Cbl transport and metabolism and their cultured cells. Figures
155-4 and 155-12 summarize the localization of these defects.
ACKNOWLEDGMENTS
The authors thank L.E. Rosenberg for his contributions to this
chapter in previous editions and R. Rozen for the provision of Fig.
155-5. We thank the many clinicians who have provided clinical
histories and ®broblasts for analysis.
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PART 17 / VITAMINS
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